Lytic Granule Release Research Articles

MADD regulates natural killer cell degranulation through Rab27a activation.

Natural killer (NK) cells have the ability to lyse other cells through the release of lytic granules (LGs). This is in part mediated by the small GTPase Rab27a, which was first identified to play a crucial role in degranulation through the study of individuals harboring mutations in the gene encoding Rab27a. However, the guanine nucleotide exchange factor (GEF) regulating the activation of Rab27a in cytotoxic lymphocytes was unknown. Here, we show that knockout of MADD significantly decreased the levels of GTP-bound Rab27a in both resting and stimulated NK cells, and MADD-deficient NK cells and CD8+ T cells displayed severely reduced degranulation and cytolytic ability, similar to that seen with Rab27a deficiency. Although MADD colocalized with Rab27a on LGs and was enriched at the cytolytic synapse, the loss of MADD did not impact Rab27a association with LGs nor their recruitment to the cytolytic synapse. Together, our results demonstrate an important role for MADD in cytotoxic lymphocyte killing.

Abstract 1815 Cancer cells inhibit the release of proinflammatory cytokines and lytic granules in Natural Killer cells through binding of deacetylated sialic acids

Abstract 1815 Cancer cells inhibit the release of proinflammatory cytokines and lytic granules in Natural Killer cells through binding of deacetylated sialic acids

Crosstalk Between NK Cell Receptors and Tumor Membrane Hsp70-Derived Peptide: A Combined Computational and Experimental Study.

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.

The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk.

Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.

Current Progress of CAR-NK Therapy in Cancer Treatment

Simple SummaryChimeric antigen receptor (CAR)-T and -natural killer (NK) therapies are promising in cancer treatment. CAR-NK therapy gains great attention due to the lack of adverse effects observed in CAR-T therapies and to the NK cells’ unique mechanisms of recognizing target cells. Off-the-shelf products are in urgent need, not only for good yields, but also for lower cost and shorter preparation time. The current progress of CAR-NK therapy is discussed.CD8+ T cells and natural killer (NK) cells eliminate target cells through the release of lytic granules and Fas ligand (FasL)-induced target cell apoptosis. The introduction of chimeric antigen receptor (CAR) makes these two types of cells selective and effective in killing cancer cells. The success of CAR-T therapy in the treatment of acute lymphoblastic leukemia (ALL) and other types of blood cancers proved that the immunotherapy is an effective approach in fighting against cancers, yet adverse effects, such as graft versus host disease (GvHD) and cytokine release syndrome (CRS), cannot be ignored for the CAR-T therapy. CAR-NK therapy, then, has its advantage in lacking these adverse effects and works as effective as CAR-T in terms of killing. Despite these, NK cells are known to be hard to transduce, expand in vitro, and sustain shorter in vivo comparing to infiltrated T cells. Moreover, CAR-NK therapy faces challenges as CAR-T therapy does, e.g., the time, the cost, and the potential biohazard due to the use of animal-derived products. Thus, enormous efforts are needed to develop safe, effective, and large-scalable protocols for obtaining CAR-NK cells. Here, we reviewed current progress of CAR-NK therapy, including its biological properties, CAR compositions, preparation of CAR-NK cells, and clinical progresses. We also discussed safety issues raised from genetic engineering. We hope this review is instructive to the research community and a broad range of readers.

Locked and Loaded: Mechanisms Regulating Natural Killer Cell Lytic Granule Biogenesis and Release.

NK cell-mediated cytotoxicity is a critical element of our immune system required for protection from microbial infections and cancer. NK cells bind to and eliminate infected or cancerous cells via direct secretion of cytotoxic molecules toward the bound target cells. In this review, we summarize the current understanding of the molecular regulations of NK cell cytotoxicity, focusing on lytic granule development and degranulation processes. NK cells synthesize apoptosis-inducing proteins and package them into specialized organelles known as lytic granules (LGs). Upon activation of NK cells, LGs converge with the microtubule organizing center through dynein-dependent movement along microtubules, ultimately polarizing to the cytotoxic synapse where they subsequently fuse with the NK plasma membrane. From LGs biogenesis to degranulation, NK cells utilize several strategies to protect themselves from their own cytotoxic molecules. Additionally, molecular pathways that enable NK cells to perform serial killing are beginning to be elucidated. These advances in the understanding of the molecular pathways behind NK cell cytotoxicity will be important to not only improve current NK cell-based anti-cancer therapies but also to support the discovery of additional therapeutic opportunities.

Ultrarapid lytic granule release from CTLs activates Ca2+-dependent synaptic resistance pathways in melanoma cells.

Human cytotoxic T lymphocytes (CTLs) exhibit ultrarapid lytic granule secretion, but whether melanoma cells mobilize defense mechanisms with commensurate rapidity remains unknown. We used single-cell time-lapse microscopy to offer high spatiotemporal resolution analyses of subcellular events in melanoma cells upon CTL attack. Target cell perforation initiated an intracellular Ca2+ wave that propagated outward from the synapse within milliseconds and triggered lysosomal mobilization to the synapse, facilitating membrane repair and conferring resistance to CTL induced cytotoxicity. Inhibition of Ca2+ flux and silencing of synaptotagmin VII limited synaptic lysosomal exposure and enhanced cytotoxicity. Multiplexed immunohistochemistry of patient melanoma nodules combined with automated image analysis showed that melanoma cells facing CD8+ CTLs in the tumor periphery or peritumoral area exhibited significant lysosomal enrichment. Our results identified synaptic Ca2+ entry as the definitive trigger for lysosomal deployment to the synapse upon CTL attack and highlighted an unpredicted defensive topology of lysosome distribution in melanoma nodules.

Natural Killer Cell Antibody-Dependent Cellular Cytotoxicity (ADCC) Activity Against Plasmodium falciparum-Infected Red Blood Cells.

Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cell defense that bridges the innate and adaptive immune systems. ADCC has been found to be a major route of immune protection against both viral infections and cancer. Recently, natural killer (NK) cell-mediated ADCC has been shown as a mechanism of parasite clearance and protection against malaria (Hart et al. J Exp Med 216(6):1280-1290, 2019). NK cells bind to antibodies on the surface of Plasmodium falciparum infected red blood cells (iRBCs) via their Fc receptor, FcγRIIIa (CD16). This interaction induces the release of lytic granules (degranulation) by NK cells, which contain perforin, granzyme B, and granulysin, as well as the secretion of IFNγ. ADCC subsequently limits the growth of Plasmodium falciparum in vitro (Arora et al. Elife:7, e36806, 2018). Because NK cell-mediated ADCC has been shown to be important in malaria parasite clearance and protection, further studies understanding ADCC in malaria are warranted (Hart et al. J Exp Med 216(6):1280-1290, 2019). Therefore, this protocol describes methods to perform an in vitro ADCC functional assay with iRBCs. Specifically, it includes protocols on how to grow and culture iRBCs, how to culture peripheral blood mononuclear cells (PMBCs), and how to do in vitro PBMC assays to measure NK degranulation and IFNγ production as measures of NK ADCC function.

The septin cytoskeleton regulates natural killer cell lytic granule release.

Natural killer (NK) cell-mediated killing involves the membrane fusion of preformed lytic granules. While the roles of actin and microtubules are well accepted during this process, the function of septins, another cytoskeletal component that associates with actin and microtubules, has not been investigated. Here we show that genetic depletion or pharmacologic stabilization of the septin cytoskeleton significantly inhibited NK cell cytotoxicity. Although the stabilization of septin filaments impaired conjugate formation, depletion of septin proteins had no impact on conjugate formation, lytic granule convergence, or MTOC polarization to the cytotoxic synapse (CS). Interestingly, septins copurify and accumulate near the polarized lytic granules at the CS, where they regulate lytic granule release. Mechanistically, we find that septin 7 interacts with the SNARE protein syntaxin 11 and facilitates its interaction with syntaxin binding protein 2 to promote lytic granule fusion. Altogether, our data identify a critical role for septins in regulating the release of lytic granule contents during NK cell-mediated killing.

Phenotypic shift of small intestinal intra-epithelial type 1 innate lymphoid cells in celiac disease is associated with enhanced cytotoxic potential

The small intestinal (SI) epithelium harbors a heterogeneous population of lymphocytes that mediate mucosal damage and repair in celiac disease (CD). The composition and roles of human proximal SI intra-epithelial innate lymphoid cells (ILCs), and their alterations in CD, are not well understood. We report that duodenal intra-epithelial ILCs predominantly consist of natural killer (NK)p44+ CD127- cytotoxic ILC1s and NKp44- CD127+ helper ILC1s, while ILC3s only represent a minor population. In patients with newly diagnosed or active CD (ACD) and refractory CD type 1 (RCD I), the frequency of SI NKp44+ ILCs is decreased, with restoration of NKp44+ ILC frequency observed in patients adhering to a gluten-free diet who show evidence of mucosal healing. Moreover, the frequency of SI NKp44- ILCs is increased in ACD and RCD I patients and correlates with the severity of villous atrophy and epithelial damage, as assessed by serum levels of fatty acid binding protein 2 (FABP2). We show that the ILC alterations in CD represent a phenotypic shift of cytotoxic ILC1s rather than an increase in helper ILC1s or transdifferentiation of ILC1sto ILC3s, and activation-induced loss of NKp44 by cytotoxic ILC1s is associated with increased interferon (IFN)-γ expression and release of lytic granules. These findings suggest that intra-epithelial NKp44- CD127- cytotoxic ILC1s may contribute to mucosal damage in CD.

NK cell detachment from target cells is regulated by successful cytotoxicity and influences cytokine production.

Natural killer (NK) cells participate in early immune defenses against pathogens and tumors and play a major role as immune effector and regulatory cells. The NK cell-mediated elimination of an infected or cancerous cell is a highly regulated process that requires the formation of a cell contact, the establishment of an immunological synapse and the polarization and release of lytic granules. Additionally, the detachment of NK cells from target cells is important for NK cells to bind and kill other cells in a process called serial killing. However, very little is known about this detachment process. Here, we show that NK detachment is directly connected to the successful killing of a target cell. The inhibition of killing due to reduced NK cell cytotoxicity or increased target cell resistance results in defective detachment and prolonged contact times. This effect leads to sustained Ca2+ flux in NK cells and the hypersecretion of proinflammatory cytokines. Linking defective cytotoxicity with enhanced cytokine secretion via reduced detachment may explain inflammatory pathologies in several diseases.

Mechanisms of natural killer cell-mediated cellular cytotoxicity.

Cellular cytotoxicity, the ability to kill other cells, is an important effector mechanism of the immune system to combat viral infections and cancer. Cytotoxic Tcells and natural killer (NK) cells are the major mediators of this activity. Here, we summarize the cytotoxic mechanisms of NKcells. NKcells can kill virally infected of transformed cells via the directed release of lytic granules or by inducing death receptor-mediated apoptosis via the expression of Fas ligand or TRAIL. The biogenesis of perforin and granzymes, the major components of lytic granules, is a highly regulated process to prevent damage during the synthesis of these cytotoxic molecules. Additionally, NKcells have developed several strategies to protect themselves from the cytotoxic activity of granular content upon degranulation. While granule-mediated apoptosis is a fast process, death receptor-mediated cytotoxicity requires more time. Current data suggest that these 2 cytotoxic mechanisms are regulated during the serial killing activity of NKcells. As many modern approaches of cancer immunotherapy rely on cellular cytotoxicity for their effectiveness, unraveling these pathways will be important to further progress these therapeutic strategies.

Abstract B190: WASP-dependent actin protrusions mechanically potentiate killing by cytotoxic T-cells

Abstract Cytotoxic T Lymphocytes (CTLs) play a central role in immune responses against intracellular pathogens and cancer. Cytotoxic responses are induced by the formation of the immunologic synapse after the recognition of the peptide-major histocompatibility complex by the T-cell receptor. Synapse formation is associated dramatic reorganization of both microtubules and filamentous actin (F-actin) within the T-cell. This promotes the directional secretion of toxic perforin and granzymes into the intercellular space, enhancing both the potency and the specificity of targeT-cell killing. We have previously demonstrated that force exertion by CTLs at the immunologic synapse was strongly correlated with the cytotoxic potential. Indeed, force exertion enhances cytotoxicity by increasing membrane tension on the target cell, which in turn promotes the pore-forming activity of secreted perforin. This correlation between applied force and biochemical responses, which we referr to as mechanopotentiation, raised the prospect that CTLs might use three-dimensional structures at the immunologic synapse to coordinate force exertion and lytic granules secretion. In the present study, we investigated the mechanisms underlying mechanopotentiation in CTLs using a combination of three-dimensional micropatterned stimulatory substrates and high-resolution imaging. Our results revealed that CTLs couple lytic granule release with the formation of highly dynamic F-actin rich protrusions. These protrusions, which are generated by the Wiskott-Aldrich Syndrome protein (WASP) and the Arp2/3 actin nucleation complex, are required for coordinating force exertion with cytolytic secretion allowing efficient killing. Our results provide insight into how cells organize mechanical output and emphasize the importance of studying complex, communicative interfaces in three dimensions. Citation Format: Fella Tamzalit, Mitchell S. Wang, Weiyang Jin, Vitaly Boyko, John M. Heddleston, Charles T. Black, Lance C. Kam, Morgan Huse. WASP-dependent actin protrusions mechanically potentiate killing by cytotoxic T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B190.

Clostridioides difficile Activates Human Mucosal-Associated Invariant T Cells.

Clostridioides difficile infection (CDI) causes severe inflammatory responses at the intestinal mucosa but the immunological mechanisms underlying CDI-related immunopathology are still incompletely characterized. Here we identified for the first time that both, non-toxigenic strains as well as the hypervirulent ribotypes RT027 and RT023 of Clostridioides difficile (formerly Clostridium difficile), induced an effector phenotype in mucosal-associated invariant T (MAIT) cells. MAIT cells can directly respond to bacterial infections by recognizing MR1-presented metabolites derived from the riboflavin synthesis pathway constituting a novel class of antigens. We confirmed functional riboflavin synthesis of C. difficile and found fixed bacteria capable of activating primary human MAIT cells in a dose-dependent manner. C. difficile-activated MAIT cells showed an increased and MR1-dependent expression of CD69, proinflammatory IFNγ, and the lytic granule components granzyme B and perforin. Effector protein expression was accompanied by the release of lytic granules, which, in contrast to other effector functions, was mainly induced by IL-12 and IL-18. Notably, this study revealed hypervirulent C. difficile strains to be most competent in provoking MAIT cell responses suggesting MAIT cell activation to be instrumental for the immunopathology observed in C. difficile-associated colitis. In conclusion, we provide first evidence for a link between C. difficile metabolism and innate T cell-mediated immunity in humans.

Ly49H-dependent IFNγ protein production by NK cells requires cytokine signaling that induces IFNγ mRNA transcription.

Abstract Natural killer (NK) cells recognize and attack target cells, including cancer and pathogen-infected cells, through a combination of activating and inhibitory signals that follow receptor–ligand interactions. NK cells play a critical role in controlling murine cytomegalovirus (MCMV) infection in C57BL/6 mice, which is dependent on interactions between the Ly49H NK cell activation receptor and its MCMV-encoded ligand m157. This interaction is not sufficient to activate NK cells and control MCMV infection. We previously reported that IL-12 and type I IFN produced early during MCMV infection induce granzyme B and perforin in NK cells, which allows them to efficiently kill virus-infected cells upon recognition of m157 through Ly49H. We now show that m157-dependent IFNγ production also requires cytokine signaling. Using quantitative PCR, the PrimeFlow RNA assay, and chemical inhibitors we found that cytokine signaling was required for IFNγ mRNA transcription, whereas signaling through Ly49H resulted in translation of IFNγ mRNA into protein. Furthermore, initial exposure of NK cell to cytokines enables them to produce IFNγ protein later in the absence of cytokines. We propose the following model: MCMV infection induces cytokine production including IFN-I and IL-12. These cytokines induce the production of cytolytic proteins and IFNγ mRNA. As a result, the NK cells are poised and ready for attack. Virus-infected cells are subsequently recognized by NK cells trough activating ligands such as m157. This activation signaling results in IFNγ production and the release of lytic granules, which results in elimination of the virus infected cells. Together these two pathways enable NK cells to swiftly attack and eradicate virus infections.

A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+ ]i (between 122 and 334nm) and [Ca2+ ]o (between 23 and 625μm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down-regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+ ]o . We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca2+ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell-shaped Ca2+ dependence with an optimum for cancer cell elimination at rather low [Ca2+ ]o (23-625μm) and [Ca2+ ]i (122-334nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca2+ ]o higher than 625μm. We tested this hypothesis in CTLs and indeed found that partial down-regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca2+ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000μm [Ca2+ ]o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146μm compared to 3 or 800μm, compatible with the Ca2+ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1-dependent Ca2+ signals enhance proliferation. We propose that a decrease of [Ca2+ ]o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation.

Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

Cytotoxic Tcells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact.

An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome

BackgroundChediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells.ObjectiveWe sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS.MethodsWe generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells.ResultsLoss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells.ConclusionThe cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention.

Human profilin 1 is a negative regulator of CTL mediated cell-killing and migration.

The actin-binding protein profilin1 (PFN1) plays a central role in actin dynamics, which is essential for cytotoxic T lymphocyte (CTL) functions. The functional role of PFN1 in CTLs, however still remains elusive. Here, we identify PFN1 as the only member of the profilin family expressed in primary human CD8+ T cells. Using in vitro assays, we find that PFN1 is a negative regulator of CTL-mediated elimination of target cells. Furthermore, PFN1 is involved in activation-induced lytic granule (LG) release, CTL migration and modulation of actin structures at the immunological synapse (IS). During CTL migration, PFN1 modulates the velocity, protrusion formation patterns and protrusion sustainability. In contrast, PFN1 does not significantly affect migration persistence and the rates of protrusion emergence and retraction. Under in vitro conditions mimicking a tumor microenvironment, we show that PFN1 downregulation promotes CTL invasion into a 3D matrix, without affecting the viability of CTLs in a hydrogen peroxide-enriched microenvironment. Highlighting its potential relevance in cancer, we find that in pancreatic cancer patients, PFN1 expression is substantially decreased in peripheral CD8+ T cells. Taken together, we conclude that PFN1 is a negative regulator for CTL-mediated cytotoxicity and may have an impact on CTL functionality in a tumor-related context.

T-Cell Receptor (TCR) Clonotype-Specific Differences in Inhibitory Activity of HIV-1 Cytotoxic T-Cell Clones Is Not Mediated by TCR Alone.

Functional analysis of T-cell responses in HIV-infected individuals has indicated that virus-specific CD8+ T cells with superior antiviral efficacy are well represented in HIV-1 controllers but are rare or absent in HIV-1 progressors. To define the role of individual T-cell receptor (TCR) clonotypes in differential antiviral CD8+ T-cell function, we performed detailed functional and mass cytometric cluster analysis of multiple CD8+ T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 epitope KK10 (KRWIILGLNK). Effective and ineffective CD8+ T-cell clones segregated based on responses to HIV-1-infected and peptide-loaded target cells. Following cognate peptide stimulation, effective HIV-specific clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained nonlytic cytokine and chemokine secretion than ineffective clones. To evaluate the TCR clonotype contribution to CD8+ T-cell function, we cloned the TCR α and β chain genes from one effective and two ineffective CD8+ T-cell clones from an elite controller into TCR-expressing lentivectors. We show that Jurkat/MA cells and primary CD8+ T cells transduced with lentivirus expressing TCR from one of the ineffective clones exhibited a level of activation by cognate peptide and inhibition of in vitro HIV-1 infection, respectively, that were comparable to those of the effective clonotype. Taken together, these data suggest that the potent antiviral capacity of some HIV-specific CD8+ T cells is a consequence of factors in addition to TCR sequence that modulate functionality and contribute to the increased antiviral capacity of HIV-specific CD8+ T cells in elite controllers to inhibit HIV infection.IMPORTANCE The greater ex vivo antiviral inhibitory activity of CD8+ T cells from elite controllers than from HIV-1 progressors supports the crucial role of effective HIV-specific CD8+ T cells in controlling HIV-1 replication. The contribution of TCR clonotype to inhibitory potency was investigated by delineating the responsiveness of effective and ineffective CD8+ T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 Gag-derived peptide, KK10 (KRWIILGLNK). KK10-stimulated "effective" CD8+ T-cell clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained cytokine and chemokine secretion than "ineffective" CD8+ T-cell clones. However, TCRs cloned from an effective and one of two ineffective clones conferred upon primary CD8+ T cells the equivalent potent capacity to inhibit HIV-1 infection. Taken together, these data suggest that other factors aside from intrinsic TCR-peptide-major histocompatibility complex (TCR-peptide-MHC) reactivity can contribute to the potent antiviral capacity of some HIV-specific CD8+ T-cell clones.