Cytotoxic Granule Exocytosis Research Articles

Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Harnessing the cytotoxic granule exocytosis to augment the efficacy of T-cell-engaging bispecific antibody therapy.

T-cell-engaging bispecific antibody (T-BsAb, also known as BiTE) therapy has emerged as a powerful therapeutic modality against multiple myeloma. Given that T-BsAb therapy redirects endogenous T cells to eliminate tumor cells, reinvigorating dysfunctional T cells may be a potential approach to improve the efficacy of T-BsAb. While various immunostimulatory cytokines can potentiate effector T-cell functions, the optimal cytokine treatment for T-BsAb therapy is yet to be established, partly due to a concern of cytokine release syndrome driven by aberrant interferon (IFN)-γ production. Here, we functionally screen immunostimulatory cytokines to determine an ideal combination partner for T-BsAb therapy. This approach reveals interleukin (IL)-21 as a potential immunostimulatory cytokine with the ability to augment T-BsAb-mediated release of granzyme B and perforin, without increasing IFN-γ release. Transcriptome profiling and functional characterization strongly support that IL-21 selectively targets the cytotoxic granule exocytosis pathway, but not pro-inflammatory responses. Notably, IL-21 modulates multiple steps of cytotoxic effector functions including upregulation of co-activating CD226 receptor, increasing cytotoxic granules, and promoting cytotoxic granule delivery at the immunological synapse. Indeed, T-BsAb-mediated myeloma killing is cytotoxic granule-dependent, and IL-21 priming significantly augments cytotoxic activities. Furthermore, in vivo IL-21 treatment induces cytotoxic effector reprogramming in bone marrow T cells, showing synergistic anti-myeloma effects in combination with T-BsAb therapy. Together, harnessing the cytotoxic granule exocytosis pathway by IL-21 may be a potential approach to achieve better responses by T-BsAb therapy.

T cell exocytosis testing provides superior sensitivity for the diagnosis of familial HLH

The rapid diagnosis of primary immunodeficiencies (PID) affecting cytotoxic lymphocyte granule exocytosis is commonly performed by evaluating NK cell surface CD107a expression after K562 challenge. NK cell exocytosis can also be induced by stimulating NK cells with P815 and anti-CD16 antibody. We have previously described how these NK tests can be adapted to study cytotoxic T cell CD107a exocytosis by gating CD3+CD8 +CD57+ cells after TCR engagement with P815 and anti-CD3 antibody. Here we present data comparing 92 patients with variants in genes affecting exocytosis function against 58 other PID patients diagnosed over 10 years from one center. NK and cytotoxic T cell exocytosis was tested on all patient samples. The anti-CD3T cell test returns superior sensitivity and specificity of 97.8% and 94.7% while the K562 NK test a sensitivity of 86.7% and specificity of 96.1%. The anti-CD 16 NK test was found comparable to the T cell test with values of 97.8% and 93.4%. The data was additionally juxtaposed against 63 patients with secondary HLH, 198 healthy adult donors, and 84 transport controls, all giving similar results of high accuracy. We further stratified the data by time taken between venipuncture and arrival at the testing laboratory and found that control samples up to 36 hours old showed comparable function to < 24 hours old samples. This is important as transporting samples from remote primary care centers often cannot be accomplished within 24 hours.While all three exocytosis tests return superior diagnostic sensitivity, we find the anti-CD3 antibody induced exocytosis of T cells and the anti-CD16 antibody induced exocytosis of NK cells more accurate than the current standard K562 cell stimulation for the diagnosis of PIDs affecting cytotoxic lymphocyte exocytosis. Evaluating T cells alongside NK cells acts as a confirmatory test and buffers against technical failures and the lack of NK cells which can be seen in lymphopenic patients. These assays with improved sensitivity may help us better understand PIDs that partially impair exocytosis.

Insights into the cellular pathophysiology of familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (fHLH) encompasses a group of rare inherited immune dysregulation disorders characterized by loss-of-function mutations in one of several genes involved in the assembly, exocytosis, and function of cytotoxic granules within CD8+ T cells and natural killer (NK) cells. The resulting defect in cytotoxicity allows these cells to be appropriately stimulated in response to an antigenic trigger, and also impairs their ability to effectively mediate and terminate the immune response. Consequently, there is sustained lymphocyte activation, resulting in the secretion of excessive amounts of pro-inflammatory cytokines that further activate other cells of the innate and adaptive immune systems. Together, these activated cells and pro-inflammatory cytokines mediate tissue damage that leads to multi-organ failure in the absence of treatment aimed at controlling hyperinflammation. In this article, we review these mechanisms of hyperinflammation in fHLH at the cellular level, focusing primarily on studies performed in murine models of fHLH that have provided insight into how defects in the lymphocyte cytotoxicity pathway mediate rampant and sustained immune dysregulation.

Synaptotagmin-like protein 1 is a potential diagnostic and prognostic biomarker in endometrial cancer based on bioinformatics and experiments

Endometrial cancer (EC) is one of the most common gynecologic malignancies. Identification of potential EC biomarkers is essential to improve the prognosis and development of therapies against EC. Synaptotagmin-like protein 1 (SYTL1), as a small GTPase Rab27 effector, mainly plays a role in vesicle trafficking and cytotoxic granule exocytosis in lymphocytes. However the role of SYTL1 in EC remains uncertain. We performed a comprehensive assessment of the relationship between SYTL1 and patient diagnosis and prognosis by analysis of EC patients’ data from TCGA. We employed the LinkedOmics and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database to analyze the biological function of SYTL1 in EC. In addition, the correlation between SYTL1 expression and its DNA methylation was performed by using cBioportal, UALCAN, TCGA Wanderer and MethSurv databases. We further assessed the link between SYTL1 and tumor-infiltrating immune cells by using gene set variation analysis (GSVA).Results We found that SYTL1 was highly expressed in EC patients and cell lines. And increased expression of SYTL1 was associated with age, clinical stage, histological type, histological grade and good overall survival (OS).SYTL1 DNA methylation is negatively associated with SYTL1 expression and UCEC patients’ OS. SYTL1 expression is closely correlated with immune infiltration. Furthermore, we carried out in vitro experiments to verify the results of bioinformatic analysis.Conclusion Our results demonstrated that the elevation of SYTL1 expression is associated with good OS and SYTL1 might be a potential diagnostic and prognostic marker in EC.

Identification of Potential Diagnostic Biomarkers From Circulating Cells During the Course of Sleep Deprivation-Related Myocardial Infarction Based on Bioinformatics Analyses

BackgroundMyocardial infarction (MI) is the leading cause of death from non-infectious diseases worldwide and results in rapid deterioration due to the sudden rupture of plaques associated with atherosclerosis, a chronic inflammatory disease. Sleep is a key factor that regulates immune homeostasis of the body. The imbalance in circulating immune cells caused by sleep deprivation (SD) may represent a risk factor leading to the rapid deterioration of plaques and MI. Therefore, it is of profound significance to identify diagnostic biomarkers for preventing SD-related MI.MethodsIn the present study, we identified coexpressed differentially expressed genes (co-DEGs) between peripheral blood mononuclear cells from MI and SD samples (compared to controls) from a public database. LASSO regression analysis was applied to identify significant diagnostic biomarkers from co-DEGs. Moreover, receiver operating characteristic (ROC) curve analysis was performed to test biomarker accuracy and diagnostic ability. We further analyzed immune cell enrichment in MI and SD samples using the CIBERSORT algorithm, and the correlation between biomarkers and immune cell composition was assessed. We also investigated whether diagnostic biomarkers are involved in immune cell signaling pathways in SD-related MI processes.ResultsA total of 10 downregulated co-DEGs from the sets of MI-DEGs and SD-DEGs were overlapped. After applying LASSO regression analysis, SYTL2, KLRD1, and C12orf75 were selected and validated as diagnostic biomarkers using ROC analysis. Next, we found that resting NK cells were downregulated in both the MI samples and SD samples, which is similar to the changes noted for SYTL2. Importantly, SYTL2 was strongly positively correlated not only with resting NK cells but also with most genes related to NK cell markers in the MI and SD datasets. Moreover, SYTL2 was highly associated with genes in NK cell signaling pathways, including the MAPK signaling pathway, cytotoxic granule movement and exocytosis, and NK cell activation. Furthermore, GSEA and KEGG analyses provided evidence that the DEGs identified from MI samples with low vs. high SYTL2 expression exhibited a strong association with the regulation of the immune response and NK cell-mediated cytotoxicity.ConclusionIn conclusion, SYTL2, KLRD1, and C12orf75 represent potential diagnostic biomarkers of MI. The association between SYTL2 and resting NK cells may be critically involved in SD-related MI development and occurrence.

Enhanced Calcium Signal Induces NK Cell Degranulation but Inhibits Its Cytotoxic Activity.

Although the mechanism of NK cell activation is still unclear, the strict calcium dependence remains the hallmark for lytic granule secretion. A plethora of studies claiming that impaired Ca2+ signaling leads to severely defective cytotoxic granule exocytosis accompanied by weak target cell lysis has been published. However, there has been little discussion about the effect of induced calcium signal on NK cell cytotoxicity. In our study, we observed that small-molecule inhibitor UNC1999, which suppresses global H3K27 trimethylation (H3K27me3) of human NK cells, induced a PKD2-dependent calcium signal. Enhanced calcium entry led to unbalanced vesicle release, which resulted into fewer target cells acquiring lytic granules and subsequently being killed. Further analyses revealed that the ability of conjugate formation, lytic synapse formation, and granule polarization were normal in NK cells treated with UNC1999. Cumulatively, these data indicated that induced calcium signal exclusively enhances unbalanced degranulation that further inhibits their cytotoxic activity in human NK cells.

Digenic Inheritance: Evidence and Gaps in Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by the inability to properly terminate an immune response. Familial HLH (FHLH) and related immune dysregulation syndromes are associated with mutations in the genes PRF1, UNC13D, STX11, STXBP2, LYST, AP3B1, and RAB27A, all of which are required for the assembly, exocytosis, and function of cytotoxic granules within CD8+ T cells and natural killer (NK) cells. Loss-of-function mutations in these genes render the cytotoxicity pathway ineffective, thereby failing to eradicate immune stimuli, such as infectious pathogens or malignant cells. The resulting persistent immune system stimulation drives hypercytokinemia, ultimately leading to severe tissue inflammation and end-organ damage. Traditionally, a diagnosis of FHLH requires the identification of biallelic loss-of-function mutations in one of these degranulation pathway genes. However, this narrow definition fails to encompass patients with other genetic mechanisms underlying degranulation pathway dysfunction. In particular, mounting clinical evidence supports a potential digenic mode of inheritance of FHLH in which single loss-of-function mutations in two different degranulation pathway genes cooperate to impair pathway activity. Here, we review the functions of the FHLH-associated genes within the degranulation pathway and summarize clinical evidence supporting a model in which cumulative defects along this mechanistic pathway may underlie HLH.

Hemophagocytic lymphohistiocytosis, what about hair?

A 1-month-old boy, born to nonconsanguineous parents, without notable family health history, presented with febrile pancytopenia and splenomegaly. Clinical examination disclosed no other anomaly, notably no neurological alteration. His initial complete blood count showed normocytic anemia (hemoglobin 105 g/L), thrombocytopenia (84 × 109/L), and neutropenia (0.8 × 109/L) without lymphopenia. Upon worsening of this pancytopenia, associated with persistent fever despite antibiotics, a bone marrow aspirate revealed no blasts but the presence of histiocytes with hemophagocytosis (Image 1A). High serum triglycerides, ferritin, and lactic dehydrogenase were observed at respectively 3.3 mmol/L, 71 000 μg/L, and 788 U/L together with hypofibrinogenemia (1.1 g/L). Molecular tests for toxoplasmosis, HSV, EBV, CMV, HHV6, and parvovirus B19 were negative. Clinical examination revealed the presence of partial albinism with silver-gray hair. This feature associated to the biological anomalies, evoked a diagnosis of Griscelli or Chediak Higashi syndrome. However, bone marrow cells did not contain giant cytoplasmic granulations. Moreover, microscopic analysis of hair revealed clumps of melanin of various sizes irregularly distributed throughout the hair shaft, suggestive of Griscelli syndrome (Image 1B,C). Cerebrospinal fluid (CSF) analysis revealed hyperproteinorachia (0.73 g/L) and 20/μL leucocytes (92% lymphocytes CD4+, CD3+, DR+, CD25−) in favor of asymptomatic central nervous system hemophagocytic lymphohistiocytosis (HLH). Sequencing of the RAB27 gene in a blood sample finally revealed a homozygous missense mutation in exon 3, confirming the diagnosis of type 2 Griscelli syndrome.1 Corticosteroid therapy was initiated followed by alemtuzumab administration. The patient then underwent a haplo-identical allogeneic hematopoietic stem cell transplantation and is well 8 months later. Children with GS2 indeed present with partial albinism, associated with hemophagocytic lymphohistiocytosis. RAB27A deficiency is responsible for a defective exocytosis of cytotoxic granules from T cells and natural killer cells, which may cause secondary immune overactivation leading to HLH. In the absence of treatment, the evolution is rapidly fatal. Although poorly codified, treatment consists in suppression of the triggering factor, control of HLH with chemotherapy or immunotherapy followed by an allo-HSCT.2 The pejorative evolution of this pathology makes its early recognition essential. Thus, children presenting with HLH must systematically benefit from a search for partial albinism. The presence of silver-gray hair makes necessary microscopic examination. The authors declare that they have no conflict of interest. ME and EC performed analyses. BLC, AH and CT treated the patient. BLC, MCB and ME wrote the manuscript.

RhoG deficiency abrogates cytotoxicity of human lymphocytes and causes hemophagocytic lymphohistiocytosis

Exocytosis of cytotoxic granules (CG) by lymphocytes is required for the elimination of infected and malignant cells. Impairments in this process underly a group of diseases with dramatic hyperferritinemic inflammation termed hemophagocytic lymphohistiocytosis (HLH). Although genetic and functional studies of HLH have identified proteins controlling distinct steps of CG exocytosis, the molecular mechanisms that spatiotemporally coordinate CG release remain partially elusive. We studied a patient exhibiting characteristic clinical features of HLH associated with markedly impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell exocytosis functions, who beared biallelic deleterious mutations in the gene encoding the small GTPase RhoG. Experimental ablation of RHOG in a model cell line and primary CTLs from healthy individuals uncovered a hitherto unappreciated role of RhoG in retaining CGs in the vicinity of the plasma membrane (PM), a fundamental prerequisite for CG exocytotic release. We discovered that RhoG engages in a protein–protein interaction with Munc13-4, an exocytosis protein essential for CG fusion with the PM. We show that this interaction is critical for docking of Munc13-4+ CGs to the PM and subsequent membrane fusion and release of CG content. Thus, our study illuminates RhoG as a novel essential regulator of human lymphocyte cytotoxicity and provides the molecular pathomechanism behind the identified here and previously unreported genetically determined form of HLH.

The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation.

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

Various Stages of Immune Synapse Formation Are Differently Dependent on the Strength of the TCR Stimulus.

Cytotoxic T lymphocytes (CTL) are key players of the adaptive immune system that target tumors and infected cells. A central step to that is the formation of a cell–cell contact zone between the CTL and its target called an immune synapse (IS). Here, we investigate the influence of the initial T cell receptor (TCR) trigger of a cytolytic IS on the distinct steps leading to cytotoxic granule (CG) exocytosis. We stimulated primary CTLs from mouse using lipid bilayers with varying anti-CD3 but constant ICAM concentrations. We fluorescently labeled molecular markers of distinct IS zones such as actin, CD3, granzyme B, and Synaptobrevin2 in CTLs and imaged cytolytic IS formation by total internal reflection fluorescence microscopy (TIRFM). We found that an intermediate anti-CD3 concentration of 10 µg/mL induces the fastest adhesion of CTLs to the bilayers and results in maximal CG fusion efficiency. The latency of actin ring formation, dwell time, and maximum surface area at the IS exhibit different dependencies on the stimulatory anti-CD3 concentrations. The number and surface area of CD3 clusters at the IS seem to show a different dependency to the TCR trigger when compared to their dwell time. Finally, the mode of full CG exocytosis appears to be independent of the TCR trigger.

Cytotoxic Granule Exocytosis From Human Cytotoxic T Lymphocytes Is Mediated by VAMP7.

Cytotoxic T lymphocytes kill infected or malignant cells through the directed release of cytotoxic substances at the site of target cell contact, the immunological synapse. While genetic association studies of genes predisposing to early-onset life-threatening hemophagocytic lymphohistiocytosis has identified components of the plasma membrane fusion machinery, the identity of the vesicular components remain enigmatic. Here, we identify VAMP7 as an essential component of the vesicular fusion machinery of primary, human T cells. VAMP7 co-localizes with granule markers throughout all stages of T cell maturation and simultaneously fuses with granule markers at the IS. Knock-down of VAMP7 expression significantly decreased the killing efficiency of T cells, without diminishing early T cell receptor signaling. VAMP7 exerts its function in a SNARE complex with Syntaxin11 and SNAP-23 on the plasma membrane. The identification of the minimal fusion machinery in T cells provides a starting point for the development of potential drugs in immunotherapy.

Cell therapy based on the chimeric receptor NKG2D

Cell therapy based on the chimeric receptor NKG2D

Effect of Natural Compounds on NK Cell Activation

Natural killer (NK) cells are lymphocytes of the innate immune system that survey the body for stressed and abnormal cells. The integration of signals that they receive through various inhibitory and activating cell surface receptors controls their activation and ability to kill target cells and produce cytokines. In this manner, phenotypically and functionally distinct subsets of NK cells help protect against microbial infections and cancer and shape the adaptive immune response. NK cells can use two different mechanisms to kill their targets, either by cytotoxic granule exocytosis or by induction of death receptor-mediated apoptosis. Death ligands belong to the tumor necrosis factor (TNF) family of ligands. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell through which granzymes and associated molecules can enter and induce apoptosis. NK cells are also involved in antibody-dependent cellular toxicity via the CD16 receptor. In addition to target recognition, NK cells can be also activated by treatment with multiple compounds with stimulatory properties. Apart from interleukins, which belong to the best characterized group of NK cell-stimulating compounds, vitamins and constituents extracted from plants also display the ability to activate NK cells. The current review characterizes several groups of NK cell-activating compounds: vitamins belonging to classes A, B, C, D, and E, polysaccharides, lectins, and a number of phytochemicals used in cancer research, exhibiting stimulatory properties when applied to NK cells. Although in most cases the exact mechanism of action is not known, constituents described in this review seem to be promising candidates for NK cell-stimulating drugs.

HPV18 E1-antigen specific CD8+ T cells show cross-reactivity against cells expressing E1 from HPV16, 31, 33 and 53.

Abstract CD8+ mediated immune response plays a major role in the clearance of Human Papillomavirus (HPV) infected cells. Although HPV vaccines prevent HPV infections through humoral response, they do not protect against established infections and its cross protection is limited. In the HPV infection, the expression of E1 helicase during viral replication makes it a potential immunological target. The E1 protein is one of the most conserved proteins among papillomaviruses. We demonstrate in previous work, that immunization of HPV type 18 E1 protein plus α-GalactosylCeramide (α-GalCer) elicited CD8 T cell-immune response, able to eliminate tumor cells expressing HPV18 E1 protein. For this study, we analyzed the amino acids sequence of E1carboxi-terminal (CT) domains from HPV18, 16, 31, 33 and 53, using Rankpep and Clustal software to determine the number of conserved peptides among E1 proteins. These sequences share 20 predicted peptides. We cloned E1-CT domains of HPV16, 31, 33 and 53, and its expression in B16-F0 cells was determine. We show that CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer, are actively cytotoxic in co-culture with B16-F0 cells expressing different E1-CT proteins in comparison with unimmunized mice, showing a cross-reactivity. Cytotoxicity activity was evaluated by detection of CD107a and CD107b on the CD8 T cells surface as part of cytotoxic granule exocytosis, through flow cytometry. Ongoing experiments will examine if mice immunized with HPV18 E1 protein plus α-GalCer are able to eliminate tumors cells expressing different E1-CT proteins in a mouse model. The characterization of new immunological targets against HPV, could help to develop new strategies to clearance HPV established infections.

Human Body Composition and Immunity: Visceral Adipose Tissue Produces IL-15 and Muscle Strength Inversely Correlates with NK Cell Function in Elderly Humans.

Natural killer (NK) lymphocyte-mediated cytotoxicity and cytokine secretion control infections and cancers, but these crucial activities decline with age. NK cell development, homeostasis, and function require IL-15 and its chaperone, IL-15 receptor alpha (IL-15Rα). Macrophages and dendritic cells (DC) are major sources of these proteins. We had previously postulated that additional IL-15 and IL-15Rα is made by skeletal muscle and adipose tissue. These sources may be important in aging, when IL-15-producing immune cells decline. NK cells circulate through adipose tissue, where they may be exposed to local IL-15. The objectives of this work were to determine (1) if human muscle, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) are sources of IL-15 and IL-15 Rα, and (2) whether any of these tissues correlate with NK cell activity in elderly humans. We first investigated IL-15 and IL-15Rα RNA expression in paired muscle and SAT biopsies from healthy human subjects. Both tissues expressed these transcripts, but IL-15Rα RNA levels were higher in SAT than in skeletal muscle. We also investigated tissue obtained from surgeries and found that SAT and VAT expressed equivalent amounts of IL-15 and IL-15Rα RNA, respectively. Furthermore, stromal vascular fraction cells expressed more IL-15 RNA than did adipocytes. To test if these findings related to circulating IL-15 protein and NK cell function, we tested 50 healthy adults aged > 70 years old. Plasma IL-15 levels significantly correlated with abdominal VAT mass in the entire cohort and in non-obese subjects. However, plasma IL-15 levels did not correlate with skeletal muscle cross-sectional area and correlated inversely with muscle strength. Plasma IL-15 did correlate with NK cell cytotoxic granule exocytosis and with CCL4 (MIP-1β) production in response to NKp46-crosslinking. Additionally, NK cell responses to K562 leukemia cells correlated inversely with muscle strength. With aging, immune function declines while infections, cancers, and deaths increase. We propose that VAT-derived IL-15 and IL-15Rα is a compensatory NK cell support mechanism in elderly humans.

Oral IL-10 suppresses colon carcinogenesis via elimination of pathogenicCD4+ T-cells and induction of antitumor CD8+ T-cell activity

ABSTRACT An oral sustained-release formulation of Interleukin-10 suppressed tumor growth and enhanced survival in the APCmin/+/Bacteroides fragilis spontaneous colon cancer model. Therapeutic benefit was associated with a 5-fold reduction in CD4+RORγt+Foxp3−IL-17+ T-helper cell, CD4+RORγt+Foxp3+IL-17+ pathogenic T-regulatory cell and CD4+RORγt−Foxp3+IL-17− conventional T-regulatory cell numbers and a concurrent 2-fold enhancement in CD8+ T-cell activity in the colon. Selective subset depletion and functional blockade studies demonstrated that at steady-state CD4+RORγt+IL-17+ T-cell subsets and CD4+Foxp3+ cTreg supported tumorigenesis, whereas CD8+ cytotoxic T-lymphocytes impeded tumor progression following IL-10 therapy. Suppression of tumor growth by CD8+ T-cells was associated with enhanced tumor infiltration and cytotoxic granule exocytosis. These findings establish the utility of oral IL-10 as a potential new therapeutic in the management of colon cancer and shed light on the cellular mechanisms that underlie its antitumor activity.

Immunization with Human Papillomavirus type-18 E1 protein induce CD8+ T cells with cytotoxic activity against cells expressing E1 from HPV-11, 33 and 53.

Abstract CD8+ mediated immune response plays a major role in the clearance of Human Papillomavirus (HPV) infected cells. Although HPV vaccines are effective in preventing HPV infections through humoral response, they fail to protect against some HPV types and established infections. In the natural history of an HPV infection, the expression of E1 helicase during viral replication makes it a potential immunological target. The early expressed E1 protein is one of the most conserved proteins among papillomaviruses. We demonstrate in previous work, that immunization of HPV type 18 E1 protein along with α-GalactosylCeramide (α-GalCer) adjuvant elicited CD8 T cell-immune response, able to eliminate tumor cells expressing HPV18 E1 protein. For this study, we compared the amino acids sequence of E1carboxi-terminal (CT) domain from HPV18, 6, 11, 16, 30, 31, 35, 45 and 53. The sequences have 60% of identity and preserve two motifs previously described for being common between many Helicase types. We cloned E1-CT domain of HPV11, HPV33 and HPV53, and its expression in B16-F0 cells was determine. We show that CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer, are actively cytotoxic in co-culture with B16-F0 cells expressing E1-CT of HPV11, HPV33 and HPV53 in comparison with unimmunized mice. Cytotoxicity activity was evaluated by detection of CD107a and CD107b on the surface of CD8 T cells as part of cytotoxic granule exocytosis. Ongoing experiments will examine if these CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer are also actively cytotoxic in presence of other E1 HPV types. The characterization of new immunological targets against HPV, could help to develop new strategies to clearance HPV established infections.

Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.