Lytic Function Research Articles

Phosphoinositide-3-Kinase Signaling in Human Natural Killer Cells: New Insights from Primary Immunodeficiency.

Human natural killer (NK) cells play a critical role in the control of viral infections and malignancy. Their importance in human health and disease is illustrated by severe viral infections in patients with primary immunodeficiencies that affect NK cell function and/or development. The recent identification of patients with phosphoinositide-3-kinase (PI3K)-signaling pathway mutations that can cause primary immunodeficiency provides valuable insight into the role that PI3K signaling plays in human NK cell maturation and lytic function. There is a rich literature that demonstrates a requirement for PI3K in multiple key aspects of NK cell biology, including development/maturation, homing, priming, and function. Here, I briefly review these previous studies and place them in context with recent findings from the study of primary immunodeficiency patients, particularly those with hyperactivating mutations in PI3Kδ signaling.

Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-perforin.

The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin's cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.

Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma.

T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

Proteomic and functional analysis identifies galectin-1 as a novel regulatory component of the cytotoxic granule machinery

Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress, there is still limited information on the molecular mechanisms implicated in target-driven degranulation, effector cell survival and composition and structure of the lytic granules. Here, using a proteomic approach we identified a panel of putative cytotoxic granule proteins, including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly, we identified galectin-1 (Gal1), an endogenous immune regulatory lectin, as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas–Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery, highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function.

Autophagic flux is required for the synthesis of triacylglycerols and ribosomal protein turnover in Chlamydomonas.

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen- or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in Chlamydomonas.

Is There Evidence for Myelin Modeling by Astrocytes in the Normal Adult Brain?

A set of astrocytic process associated with altered myelinated axons is described in the forebrain of normal adult rodents with confocal, electron microscopy, and 3D reconstructions. Each process consists of a protuberance that contains secretory organelles including numerous lysosomes which polarize and open next to disrupted myelinated axons. Because of the distinctive asymmetric organelle distribution and ubiquity throughout the forebrain neuropil, this enlargement is named paraxial process (PAP). The myelin envelope contiguous to the PAP displays focal disruption or disintegration. In routine electron microscopy clusters of large, confluent, lysosomes proved to be an effective landmark for PAP identification. In 3D assemblies lysosomes organize a series of interconnected saccules that open up to the plasmalemma next to the disrupted myelin envelope(s). Activity for acid hydrolases was visualized in lysosomes, and extracellularly at the PAP-myelin interface and/or between the glial and neuronal outer aspects. Organelles in astrocytic processes involved in digesting pyknotic cells and debris resemble those encountered in PAPs supporting a likewise lytic function of the later. Conversely, processes entangling tripartite synapses and glomeruli were devoid of lysosomes. Both oligodendrocytic and microglial processes were not associated with altered myelin envelopes. The possible roles of the PAP in myelin remodeling in the context of the oligodendrocyte-astrocyte interactions and in the astrocyte's secretory pathways are discussed.

Mutational analysis of the MS2 lysis protein L.

Small single-stranded nucleic acid phages effect lysis by expressing a single protein, the amurin, lacking muralytic enzymatic activity. Three amurins have been shown to act like 'protein antibiotics' by inhibiting cell-wall biosynthesis. However, the L lysis protein of the canonical ssRNA phage MS2, a 75 aa polypeptide, causes lysis by an unknown mechanism without affecting net peptidoglycan synthesis. To identify residues important for lytic function, randomly mutagenized alleles of L were generated, cloned into an inducible plasmid and the transformants were selected on agar containing the inducer. From a total of 396 clones, 67 were unique single base-pair changes that rendered L non-functional, of which 44 were missense mutants and 23 were nonsense mutants. Most of the non-functional missense alleles that accumulated in levels comparable to the wild-type allele are localized in the C-terminal half of L, clustered in and around an LS dipeptide sequence. The LS motif was used to align L genes from ssRNA phages lacking any sequence similarity to MS2 or to each other. This alignment revealed a conserved domain structure, in terms of charge, hydrophobic character and predicted helical content. None of the missense mutants affected membrane-association of L. Several of the L mutations in the central domains were highly conservative and recessive, suggesting a defect in a heterotypic protein-protein interaction, rather than in direct disruption of the bilayer structure, as had been previously proposed for L.

Functional plasticity of human Tc17 cells is dependent on cytokine milieu

Abstract Human Tc17 cells (IL-17-producing CD8+ T cells) have been identified, but little is known about their functional characteristics and mechanism of their plasticity. We aimed to optimize the differentiation of Tc17 cells and then investigate whether the reprograming of Tc17 to Tc1 cells can promote the anti-tumor cytotoxicity. In this study, we isolated CD8+ T cells from the peripheral blood of healthy donors to induce Tc17 differentiation. Human CD8+ T cells were stimulated with anti-CD3/anti-CD28 antibodies, TGF-β, IL-1β, IL-6, IL-23, IL-2, anti-IFN-γ, and anti-IL-4 antibodies. After 10 days in culture, Tc17 cells were about 10% IL-17+. Although Tc17 cells had upregulated anti-apoptotic proteins, the expression of cytotoxic molecules and lytic function of Tc17 cells were weaker than Tc1 cells. Interestingly, Tc17 cells could be converted to Tc1 cells under the stimulation of specific cytokine milieu, and their lytic function improved after conversion. Given these findings of significant enhancement of cytotoxicity, we suggest that reprogramming of human Tc17 cells can be used as an immunotherapeutic strategy against cancer.

Signaling Pathways Involved in Tributyltin‐Induced Increases in Interleukin 6 Production by Lymphocytes

Tributyltin (TBT) is a toxic compound that has industrial uses, such as slime control on masonry, wood preservation, and disinfection of circulating industrial cooling water and is most frequently used in antifouling paints on the hulls of ships. As a result of TBT's lipophilic character, it absorbs readily into organisms and contaminates the environment and is found in human blood in concentrations as high as 261 nM. TBT decreases the lytic function and secretion of interferon gamma (IFNγ), tumor necrosis factor alpha and interleukin 1 beta (IL‐1β) from human lymphocytes. IL‐6 is a cytokine which regulates the function of many cells including tumor cells. Recent studies in our lab have shown that TBT alters production of IL‐6 by lymphocytes. The current study aims to determine whether TBT utilizes MAPK signaling pathways (ERK 1/2, p38) to cause alterations in IL‐6 production. TBT‐induced production of IL‐6 in human lymphocytes was measured in the presence and absence of ERK1/2 pathway (PD98059) and p38 (SB202190). Results indicated that the stimulation of IL‐6 production by TBT decreased in the presence of each of the MAPK pathway inhibitors. These data suggest that TBT may be causing increased IL‐6 production by activating ERK1/2 and p38 MAPKs.Support or Funding InformationSupported by NIH grant 5U54CA163066

CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function.

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β–T cells (TCRβ–null) are highly susceptible and die over 10–18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

Effects of pentachlorophenol and dichlorodiphenyltrichloroethane on secretion of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) from human immune cells

Pentachlorophenol (PCP) and dichlorodiphenyltrichloroethane (DDT) are pesticides that have been widely used and significantly contaminate the environment. Both are found in human blood and have been shown to alter the lytic and binding function of human natural killer (NK) cells. Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) are pro-inflammatory cytokines, which regulate immune responsiveness to pathogens and tumors. Their levels require very tight control to prevent loss of immune competence or excessive inflammation. Here, we examined the capacity of PCP and DDT to alter the secretion of these critical pro-inflammatory cytokines from increasingly reconstituted (more complex) preparations of human immune cells which included NK cells, monocyte-depleted (MD) peripheral blood mononuclear cells (PBMCs) (a preparation that is predominantly lymphocytes) and PBMCs (a preparation containing lymphocytes and monocytes). Results indicated that exposure to PCP decreased IFNγ secretion at the highest exposures (2.5 and 5 μM) and increased IFNγ secretion at lower concentrations. These effects were seen irrespective of the complexity of the cell preparation. PCP at 2.5 and 5 μM generally decreased TNFα secretion from NK cells, but had inconsistent effects in MD-PBMCs and PBMCs. Exposure of each of the immune cell preparations to DDT caused increase in IFNγ secretion. DDT (2.5 μM) increased TNFα secretion from MD-PBMCs after either 24 h or 48 h of exposure. The mechanism of PCP-induced increase in IFNγ secretion appears to involve the p38 mitogen activated protein kinase (MAPK) pathway, based on loss of PCP stimulated increase when this pathway was inhibited.

Exposures to the environmental toxicants pentachlorophenol (PCP) and dichlorodiphenyltrichloroethane (DDT) modify secretion of interleukin 1-beta (IL-1β) from human immune cells.

Pentachlorophenol (PCP) and Dichlorodiphenyltrichloroethane (DDT) are environmental contaminants found in human blood. Previous studies have shown that PCP and DDT inhibit the lytic function of highly purified human natural killer (NK) lymphocytes and decrease the expression of several surface proteins on NK cells. Interleukin-1 βeta (IL-1β) is a cytokine produced by lymphocytes and monocytes, and anything that elevates its levels inappropriately can lead to chronic inflammation, which among other consequences can increase tumor development and invasiveness. Here, PCP and DDT were examined for their ability to alter secretion of IL-1β from immune cell preparations of various complexity: NK cells; monocyte-depleted (MD) peripheral blood mononuclear cells (PBMCS); and PBMCs. Cells were exposed to concentrations of PCP ranging from 5 to 0.05µM and DDT concentrations of 2.5-0.025μM for 24, 48h, and 6days. Results showed that both PCP and DDT increased IL-1β secretion from all of the immune cell preparations. The specific concentrations of PCP and DDT that increased IL-1β secretion varied by donor. Immune cells from all donors showed compound-induced increases in IL-1β secretion at one or more concentration at one or more length of exposure. The mechanism of PCP stimulation of IL1-β secretion was also addressed, and it appears that the MAPKs, ERK1/2 and p38, may be utilized by PCP to stimulate secretion of IL-1β.

Recombinant Poxvirus and the Tumor Microenvironment: Oncolysis, Immune Regulation and Immunization

Oncolytic viruses (OVs) are being extensively studied for their potential roles in the development of cancer therapy regimens. In addition to their direct lytic effects, OVs can initiate and drive systemic antitumor immunity indirectly via release of tumor antigen, as well as by encoding and delivering immunostimulatory molecules. This combination makes them an effective platform for the development of immunotherapeutic strategies beyond their primary lytic function. Engineering the viruses to also express tumor-associated antigens (TAAs) allows them to simultaneously serve as therapeutic vaccines, targeting and amplifying an immune response to TAAs. Our group and others have shown that vaccinating intratumorally with a poxvirus that encodes TAAs, in addition to immune stimulatory molecules, can modulate the tumor microenvironment, overcome immune inhibitory pathways, and drive both local and systemic tumor specific immune responses.

762. A Quantitative Imaging Tool Box for the Functional Analysis of Chimeric Antigen Receptor - T Cell Immune Synapse Helps Identify Novel Structural and Functional Features of CAR T Cells

Introduction: The cytolytic immunological synapse (IS) in T cells is a discrete structural entity formed after the ligation of specific activation receptors and leading to the destruction of a cancerous cell through the targeted release of the contents of T cell lytic granules. Although the “native” T cell IS formation where the TCR binds to antigenic peptide-MHC complex has been intensely studied, the “engineered” CAR T cell synapse and its variations based on single chain variable fragment (scFv) design and co-stimulatory domain arrangements, still remains a black box. Our work focuses on the quantitative evaluation of the CAR T cell lytic IS using highly quantitative high and super resolution imaging techniques. Specifically, by combining quantitative interrogations of CAR synapse formation kinetics and dynamics at the cellular level, we are able to obtain an unprecedented level of insight into the effectiveness of therapy cells relative to the traditional measures of cytotoxicity and effector assays like chromium release, ELISPOT and flow cytometry. Super-resolution microscopy enables us to further “see” subtle variations in therapy cell quality at a near molecular level and can be used as a very precise functional readout of CAR T cell rational design. Methods: High and super resolution fluorescence microscopy platforms are used to image CAR T cell-target conjugates in the x, y and z dimensions. Imaging parameters include direct evaluation of CAR engagement at the IS by measuring mean fluorescence intensity (MFI) and volume of synaptic aggregates of CAR specific ligands as well as their relative distribution at the synapse. Results: We report here the quantitative measurement and comparison of single and dual CAR targeted ligand aggregation at the IS, lytic machinery re-arrangement and time to target cell killing. Our studies using this predictive imaging tool box establishes the superiority of T cells expressing a bispecific (tandem) CAR to those expressing one or two monospecific CARs by the presence of significantly increased accumulation of F-Actin (> five fold), greater convergence and polarization of the lytic granule machinery. Dual engagement of the targeted ligands by a tandem CAR also significantly offsets antigen escape variants in tumors predicting reduced tumor relapse. Furthermore, application of our imaging tool box reveals that tonic signaling of CARs containing a 4-1BB co-stimulatory domain can result in an almost three fold upregulation of Fas on plasma membrane and co-localization with Fas L leading to enhanced apoptosis and lower persistence of CAR T cells. Conclusions: Using our quantitative imaging tool box we are able to define a specific list of parameters to measure CAR engagement and lytic function at the IS. These imaging parameters can be directly applied to interrogate and predict functionality outcomes upon CAR design variations. By doing this we hope to provide crucial early insight in CAR T cell design that can guide improvements in the efficacy, safety and, ultimately, clinical effectiveness of CAR T cell therapies.

Role of ERK1/2 and p38 MAPKs in Tributyltin‐stimulated Interleukin 1 Beta Secretion and Production from Human Immune Cells.

Tributyltin (TBT) is an organotin compound that contaminates the environment due to its use as a biocide in a wide variety of applications. TBT has been found in human blood samples at levels as high as 265 nM. Previously, we have demonstrated that TBT interferes with the lytic function of human natural killer lymphocytes and alters the secretion of several pro‐inflammatory cytokines from lymphocytes, including interleukin 1 beta (IL‐1β). IL‐1β regulates cellular growth and immune responsiveness. Elevated levels of IL‐1β have been shown to increase the invasiveness of tumors. Our recent studies showed that TBT exposures of 5–25 nM (levels that are seen in human blood samples) caused increased secretion of IL‐1β from monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs). In this study we examine the roles of the ERK1/2 and p38 MAPK pathways in TBT‐induced increases in IL‐1β secretion, protein production, and mRNA levels. MD‐PBMCs were treated with the MEK1/2 inhibitor PD98059 or the p38 inhibitor SB202190 for 1 h prior to exposure to TBT concentrations of 25, 10, and 5 nM for 24 h. Secretion of IL‐1β was measured using ELISA, intracellular protein levels were monitored using western blot, and mRNA expression was determined by RT‐qPCR. Results indicated that the TBT‐induced secretion of IL‐1β requires the ERK1/2 pathway. The increases in secretion are accompanied by increased protein levels in TBT‐exposed cells and this increase in protein production also utilized the ERK1/2 pathway. These data suggest that increased protein production accompanies the increases in secretion of IL‐1β seen in human immune cells exposed to TBT and these increases are dependent on MAPK pathway activation.Support or Funding InformationSupported by NIH grant 5U54CA163066.

Activation of protein kinase C and protein kinase D in human natural killer cells: effects of tributyltin, dibutyltin, and tetrabromobisphenol A

Up to now, the ability of target cells to activate protein kinase C (PKC) and protein kinase D (PKD) (which is often a downstream target of PKC) has not been examined in natural killer (NK) lymphocytes. Here we examined whether exposure of human NK cells to lysis sensitive tumor cells activated PKC and PKD. The results of these studies show for the first time that activation of PKC and PKD occurs in response to target cell binding to NK cells. Exposure of NK cells to K562 tumor cells for 10 and 30 min increased phosphorylation/activation of both PKC and PKD by roughly 2-fold. Butyltins (tributyltin (TBT), dibutyltin (DBT)) and brominated compounds (tetrabromobisphenol A (TBBPA)) are environmental contaminants that are found in human blood. Exposures of NK cells to TBT, DBT, or TBBPA decrease NK cell lytic function in part by activating the mitogen-activated protein kinases (MAPKs) that are part of the NK lytic pathway. We established that PKC and PKD are part of the lytic pathway upstream of MAPKs and thus we investigated whether DBT, TBT, and TBBPA exposures activated PKC and PKD. TBT-activated PKC by 2–3-folds at 10 min at concentrations ranging from 50 to 300 nM while DBT caused a 1.3-fold activation at 2.5 µM at 10 min. Both TBT and DBT caused an approximately 2-fold increase in phosphorylation/activation of PKC. Exposures to TBBPA caused no statistically significant changes in either PKC or PKD activation.

Umbilical cord blood plasma contains soluble NKG2D ligands that mediate loss of natural killer cell function and cytotoxicity.

NK cells play a key role in innate elimination of virally infected or neoplastic cells but they can be circumvented by immunoevasive mechanisms enabling viral spread or tumor progression. Engagement of the NKG2D activating receptor with soluble forms of its ligand is one such mechanism of inducing NK cell hyporesponsiveness. Interestingly, this immunoevasive strategy among others is described at the maternal-fetal interface where tolerance of the semi-allogeneic fetus is required to allow successful human pregnancy. Understanding of maternal-fetal tolerance is increasing but mechanisms preventing alloreactivity of fetal immune cells against the maternal host are less well understood. The study of umbilical cord blood has enabled insight of the fetal immune system, which appears immature and inert. We have found that soluble NKG2D ligands (sNKG2DLs) are present in cord blood plasma (CBP) and associate with adult NK cell hyporesponsiveness demonstrated by reduced CD107a expression and secretion of IFN-γ upon stimulation. The capacity of NK cells to kill K562 cells or proliferate was also reduced by incubation with CBP; however, physical removal of sNKG2DL from CBP restored K562 lytic function and NKG2D expression. Therefore, our results strongly suggest sNKG2DLs are expressed in CBP as a mechanism of fetal-maternal tolerance in human pregnancy.

Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor–expressing T cells

AbstractT cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ+ AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ+ THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34+ HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA.

Progression to AIDS in SIV-Infected Rhesus Macaques is Associated with Distinct KIR and MHC class I Polymorphisms and NK Cell Dysfunction.

Killer cell immunoglobulin-like receptors (KIR) regulate the activity of natural killer (NK) cells and have been shown to be associated with susceptibility to a number of human infectious diseases. Here, we analyzed NK cell function and genetic associations in a cohort of 52 rhesus macaques experimentally infected with SIVmac and subsequently stratified into high viral load (HVL) and low viral load (LVL) plasma viral loads at set point. This stratification coincided with fast (HVL) and slow (LVL) disease progression indicated by the disease course and critical clinical parameters including CD4+ T cell counts. HVL animals revealed sustained proliferation of NK cells but distinct loss of peripheral blood NK cell numbers and lytic function. Genetic analyses revealed that KIR genes 3DL05, 3DS05, and 3DL10 as well as 3DSW08, 3DLW03, and 3DSW09 are correlated, most likely due to underlying haplotypes. SIV-infection outcome associated with presence of transcripts for two inhibitory KIR genes (KIR3DL02, KIR3DL10) and three activating KIR genes (KIR3DSW08, KIR3DS02, KIR3DS05). Presence of KIR3DL02 and KIR3DSW08 was associated with LVL outcome, whereas presence of KIR3DS02 was associated with HVL outcome. Furthermore, we identified epistasis between KIR and MHC class I alleles as the transcript presence of the correlated genes KIR3DL05, KIR3DS05, and KIR3DL10 increased HVL risk when Mamu-B*012 transcripts were also present or when Mamu-A1*001 transcripts were absent. These genetic associations were mirrored by changes in the numbers, the level of proliferation, and lytic capabilities of NK cells as well as overall survival time and gastro-intestinal tissue viral load.

Synaptic release of CCL5 storage vesicles triggers CXCR4 surface expression promoting CTL migration in response to CXCL12.

The lytic function of CTL relies on the polarized release of cytotoxic granules (CG) at the immune synapse (IS) with target cells. CTL also contain CCL5 in cytoplasmic storage vesicles (CCL5V) distinct from CG, the role of which, in regulating T cell effector functions, is not understood. Using human CD8(+) T cells specific to a lung tumor-associated Ag, we show in this article that CTL release both secretory compartments into the immune synapse with autologous tumor cells. Moreover, we demonstrate that disorganization of the T cell microtubule cytoskeleton and defects in hMunc13-4 or Rab27a abrogate CG exocytosis and synaptic secretion of the chemokine. Mechanistically, synaptic release of CCL5 cytoplasmic storage vesicles likely occurs upon their coalescence with the Rab27a-hMunc13-4 compartment and results in autocrine, CCR5-dependent induction of CXCR4 cell surface expression, thereby promoting T cell migration in response to CXCL12. We propose that CCL5 polarized delivery represents a mechanism by which CTL control immune synapse duration.