Knocking 'em Dead: Pore-Forming Proteins in Immune Defense.

Immune cells use a variety of membrane-disrupting proteins [complement, perforin, perforin-2, granulysin, gasdermins, mixed lineage kinase domain-like pseudokinase (MLKL)] to induce different kinds of death of microbes and host cells, some of which cause inflammation. After activation by proteolytic cleavage or phosphorylation, these proteins oligomerize, bind to membrane lipids, and disrupt membrane integrity. These membrane disruptors play a critical role in both innate and adaptive immunity. Here we review our current knowledge of the functions, specificity, activation, and regulation of membrane-disrupting immune proteins and what is known about the mechanisms behind membrane damage, the structure of the pores they form, how the cells expressing these lethal proteins are protected, and how cells targeted for destruction can sometimes escape death by repairing membrane damage.

Granzyme B Disrupts Central Metabolism and Protein Synthesis in Bacteria to Promote an Immune Cell Death Program.

Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack.

The extracellular matrix in solid tumor immunotherapy.

The extracellular matrix (ECM) of solid tumors impacts the antitumor activities of CD8+ T and natural killer (NK) cells in a variety of ways. Cell motility is restricted by the tumor ECM which creates physical barriers. The tumor ECM directly alter the phenotypes and functions of cytotoxic lymphocytes, and indirectly influences immunological synapse-mediated interactions between cytotoxic lymphocytes and cancer cells. Therefore, strategies to improve solid tumor immunotherapy should be established by considering complex ternary interactions between cytotoxic lymphocytes, cancer cells, and the tumor ECM. Novel bioengineering tools approximating key characteristics of the tumor ECM, such as in vitro reconstituted 3D ECMs and microfluidics are valuable from a fundamental study viewpoint and from a translational perspective, aiming to enable systematic screening approaches.

Early B cell factor 4 modulates FAS-mediated apoptosis and promotes cytotoxic function in human immune cells.

Apoptosis is a genetically regulated program of cell death that plays a key role in immune disease processes. We identified EBF4, a little-studied member of the early B cell factor (EBF) family of transcription factors, in a whole-genome CRISPR screen for regulators of Fas/APO-1/CD95-mediated T cell death. Loss of EBF4 increases the half-life of the c-FLIP protein, and its presence in the Fas signaling complex impairs caspase-8 cleavage and apoptosis. Transcriptome analysis revealed that EBF4 regulates molecules such as TBX21, EOMES, granzyme, and perforin that are important for human natural killer (NK) and CD8+ T cell functions. Proximity-dependent biotin identification (Bio-ID) mass spectrometry analyses showed EBF4 binding to STAT3, STAT5, and MAP kinase 3 and a strong pathway relationship to interleukin-2 regulated genes, which are known to govern cytotoxicity pathways. Chromatin immunoprecipitation and DNA sequencing analysis defined a canonical EBF4 binding motif, 5'-CCCNNGG/AG-3', closely related to the EBF1 binding site; using a luciferase-based reporter, we found a dose-dependent transcriptional response of this motif to EBF4. We also conducted assay for transposase-accessible chromatin sequencing in EBF4-overexpressing cells and found increased chromatin accessibility upstream of granzyme and perforin and in topologically associated domains in human lymphocytes. Finally, we discovered that the EBF4 has basal expression in human but not mouse NK cells and CD8+ T cells and vanishes following activating stimulation. Together, our data reveal key features of a previously unknown transcriptional regulator of human cytotoxic immune function.

Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients.

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

EBV-positive pyothorax-associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma associated with chronic inflammation (DLBCL-CI) develops in the setting of long-standing inflammation. This type of lymphoma may have specific expression profiles of chemokines involved in the pathogenesis of DLBCL-CI. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and represents a valuable model for the study of this disease category. Using a panel of PAL cell lines, we found that PAL cells expressed and secreted C-X-C motif chemokine ligands 9 and 10 (CXCL9 and CXCL10), the ligands of CXCR3, in contrast to EBV-negative DLBCL cell lines, which did not. Culture supernatants from PAL cell lines attracted CXCR3-expressing CD4+ T cells, CD8+ T cells, and CD56+ natural killer cells from human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CXCR3-positive cytotoxic lymphocytes that expressed interferon-γ. The expression of CXCL9 and CXCL10 was detected in PAL tumor biopsy samples from patients, and CXCR3-positive lymphocytes were abundant in the tissue samples. Collectively, these findings suggest that CXCL9 and CXCL10 are produced by PAL cells and can elicit cytotoxic responses via CXCR3. This chemokine system is also likely to contribute to tissue necrosis, which is a signature histological feature of DLBCL-CI. Further studies are warranted to determine whether the CXCL9-CXCL10/CXCR3 axis exerts antitumor effects in DLBCL-CI.

Preventive effect of tertiary lymphoid structures on lymph node metastasis of lung adenocarcinoma.

BACKGROUND: Ectopic lymphoid formations are called tertiary lymphoid structures (TLSs). TLSs in cancer have been reported to be associated with good prognosis and immunotherapy response. However, the relationship between TLSs and lymph node (LN) metastasis is unclear. METHODS: We analyzed 218 patients with radically resected lung adenocarcinoma. TLSs were defined as the overlap of T cell zone and B cell zone. Granzyme B + cells were defined as cytotoxic lymphocytes. We evaluated phenotypes of lymphocytes in TLSs, tumor-infiltrating lymphocytes (TILs) and LNs by immunohistochemistry. We divided the patients into mature TLS (DC-Lamp high) and immature TLS (DC-Lamp low) groups. The relationship between TLS maturation and clinicopathological factors was analyzed. RESULTS: The mature TLS group was associated with significantly lower frequency of LN metastasis (P < 0.0001) and early cancer stage (P = 0.0049). The mature TLS group had significantly more CD8 + (P = 0.0203) and Foxp3 + (P = 0.0141) cells in TILs than the immature TLS group had. Mature TLSs were independently associated with a favorable overall survival (hazard ratio [HR] = 0.17, P = 0.0220) and disease-free survival (HR = 0.54, P = 0.0436). Multivariate analysis showed that mature TLS was an independent low-risk factor for LN metastasis (odds ratio = 0.06, P = 0.0003). The number of cytotoxic lymphocytes in LNs was higher in the mature TLS group than in the immature group (20.0 vs. 15.1, P = 0.017). CONCLUSION: Mature TLSs were associated with an increased number of cytotoxic lymphocytes in draining LNs, a lower frequency of LN metastasis, and favorable outcomes. Mature TLSs may support antitumor immunity by lymphocyte activation.

Capability of Human Dendritic Cells Pulsed with Autologous Induced Pluripotent Stem Cell Lysate to Induce Cytotoxic T Lymphocytes against HLA-A33-Matched Cancer Cells.

Irradiated murine induced-pluripotent stem cells (iPSCs) elicit the antitumor response in vivo. However, it is unclear whether human iPSCs would elicit antitumor effects. In the present study, we investigated the capability of human iPSC lysate (iPSL)-pulsed dendritic cells (DCs) (iPSL/DCs) to induce cancer-responsive cytotoxic T lymphocytes (CTLs) in vitro. iPSCs and DCs were induced from peripheral blood mononuclear cells isolated from a human leukocyte antigen (HLA)-A33 homozygous donor. The iPSL was pulsed with immature DCs, which were then stimulated to allow full maturation. The activated DCs were co-cultured with autologous CTLs and their responses to SW48 colorectal carcinoma cells (HLA-A32/A33), T47D breast cancer cells (HLA-A33/A33), and T98G glioblastoma cells (HLA-A02/A02) were tested with enzyme-linked immunospot (ELISPOT) assays. Comprehensive gene expression analysis revealed that the established iPSCs shared numerous tumor-associated antigens with the SW48 and T47D cells. Immunofluorescent analysis demonstrated that the fluorescent-labeled iPSL was captured by the immature DCs within 2 h. iPSL/DCs induced sufficient CTL numbers in 3 weeks for ELISPOT assays, which revealed that the induced CTLs responded to SW48 and T47D cells. Human iPSL/DCs induced cancer-responsive CTLs on HLA-A33-matched cancer cells in vitro and could be a promising universal cancer vaccine for treating and preventing cancer.

CD8+ T Cells Impact Rising PSA in Biochemically Relapsed Cancer Patients Using Immunotherapy Targeting Tumor-Associated Antigens.

The management of men with prostate cancer (PCa) with biochemical recurrence following local definitive therapy remains controversial. Early use of androgen deprivation therapy (ADT) leads to significant side effects. Developing an alternative, clinically effective, and well-tolerated therapy remains an unmet clinical need. INO-5150 is a synthetic DNA therapy that includes plasmids encoding for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA), and INO-9012 is a synthetic DNA plasmid encoding for interleukin-12 (IL-12). This phase 1/2, open-label, multi-center study enrolled men with PCa with rising PSA after surgery and/or radiation therapy. Patients were enrolled into one of four treatment arms: arm A, 2 mg of INO-5150; arm B, 8.5 mg of INO-5150; arm C, 2 mg of INO-5150 + 1 mg of INO-9012; and arm D, 8.5 mg of INO-5150 + 1 mg of INO-9012. Patients received study drug with electroporation on day 0 and on weeks 3, 12, and 24, and they were followed for up to 72 weeks. Sixty-two patients were enrolled. Treatment was well tolerated. 81% (50/62) of patients completed all visits. 85% (53/62) remained progression-free at 72 weeks. PSA doubling time (PSADT) was increased when assessed in patients with day 0 PSADT ≤12 months. Immunogenicity was observed in 76% (47/62) of patients by multiple assessments. Analysis indicated that CD38 and perforin co-positive CD8 T cell frequency correlated with attenuated PSA rise (p = 0.05, n = 50).

Small Molecular Immune Modulators as Anticancer Agents.

After decades of intense effort, immune checkpoint inhibitors have been conclusively demonstrated to be effective in cancer treatments and thus are revolutionizing the concepts in the treatment of cancers. Immuno-oncology has arrived and will play a key role in cancer treatment in the foreseeable future. However, efforts to find novel methods to improve the immune response to cancer have not ceased. Small-molecule approaches offer inherent advantages over biologic immunotherapies since they can cross cell membranes, penetrate into tumor tissue and tumor microenvironment more easily, and are amenable to be finely controlled than biological agents, which may help reduce immune-related adverse events seen with biologic therapies and provide more flexibility for the combination use with other therapies and superior clinical benefit. On the one hand, small-molecule therapies can modulate the immune response to cancer by restoring the antitumor immunity, promoting more effective cytotoxic lymphocyte responses, and regulating tumor microenvironment, either directly or epigenetically. On the other hand, the combination of different mechanisms of small molecules with antibodies and other biologics demonstrated admirable synergistic effect in clinical settings for cancer treatment and may expand antibodies' usefulness for broader clinical applications. This chapter provides an overview of small-molecule immunotherapeutic approaches either as monotherapy or in combination for the treatment of cancer.

Active fractions of mannoproteins derived from yeast cell wall stimulate innate and acquired immunity of adult and elderly dogs.

Nutritional intervention in older dogs aims to increase lifespan and improve life quality as well as delay the development of diseases related to ageing. It is believed that active fractions of mannoproteins (AFMs) obtained through extraction and fractionation of yeast cell walls (Saccharomyces cerevisiae) may beneficially modulate the immune system. However, studies that have evaluated this component and the effects of ageing on the immune system of dogs are scarce. This study aimed to evaluate the immunological effects of AFMs in adult and elderly dogs. Three extruded iso-nutrient experimental diets were formulated: without addition of AFM (T0); with AFM at 400 mg/kg (T400); and with AFM at 800 mg/kg (T800). Thirty-six beagle dogs were used, and six experimental treatments, resulting in combinations of age (adult and elderly) and diet (T0, T400, and T800), were evaluated. On days zero, 14, and 28, blood samples were obtained for leucocyte phenotyping and phagocytosis assays. On days zero and 28, a lymphoproliferation test, quantification of reactive oxygen (H2O2) and nitrogen (NO) intermediate production, evaluation of faecal immunoglobulin A (IgA) content, and a delayed cutaneous hypersensitivity test (DCHT) were performed. Statistical analyses were performed with SAS software. Repeated measure variance analyses were performed, and means were compared by the Tukey test. Values of P ≤ 0.05 were considered significant, and values of P ≤ 0.10 were considered tendencies. Dogs fed T400 tended to have higher neutrophilic phagocytic activity than dogs fed T800 (P = 0.073). Regarding reactive oxygen intermediates, bacterial lipopolysaccharide (LPS)-stimulated neutrophils from animals that were fed T400 had a tendency to produce more H2O2 than those from animals fed the control diet (P = 0.093). Elderly dogs, when compared to adult dogs, had lower absolute T and B lymphocyte counts, lower auxiliary T lymphocyte counts, and higher cytotoxic T lymphocyte counts (P < 0.05). A significant effect of diet, age, and time with saline inoculation was noted for the DCHT. There was no effect of diet or age on faecal IgA content in dogs. This study suggests beneficial effects of mannoproteins on the specific and nonspecific immune responses in adult and elderly dogs.

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

BACKGROUND: Chediak-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. OBJECTIVE: We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. METHODS: We 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. RESULTS: Loss 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. CONCLUSION: The 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.

Chediak-Higashi syndrome: Lysosomal trafficking regulator domains regulate exocytosis of lytic granules but not cytokine secretion by natural killer cells.

BACKGROUND: Mutations in lysosomal trafficking regulator (LYST) cause Chediak-Higashi syndrome (CHS), a rare immunodeficiency with impaired cytotoxic lymphocyte function, mainly that of natural killer (NK) cells. Our understanding of NK cell function deficiency in patients with CHS and how LYST regulates lytic granule exocytosis is very limited. OBJECTIVE: We sought to delineate cellular defects associated with LYST mutations responsible for the impaired NK cell function seen in patients with CHS. METHODS: We analyzed NK cells from patients with CHS with missense mutations in the LYST ARM/HEAT (armadillo/huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) or BEACH (beige and Chediak-Higashi) domains. RESULTS: NK cells from patients with CHS displayed severely reduced cytotoxicity. Mutations in the ARM/HEAT domain led to a reduced number of perforin-containing granules, which were significantly increased in size but able to polarize to the immunologic synapse; however, they were unable to properly fuse with the plasma membrane. Mutations in the BEACH domain resulted in formation of normal or slightly enlarged granules that had markedly impaired polarization to the IS but could be exocytosed on reaching the immunologic synapse. Perforin-containing granules in NK cells from patients with CHS did not acquire certain lysosomal markers (lysosome-associated membrane protein 1/2) but were positive for markers of transport vesicles (cation-independent mannose 6-phosphate receptor), late endosomes (Ras-associated binding protein 27a), and, to some extent, early endosomes (early endosome antigen 1), indicating a lack of integrity in the endolysosomal compartments. NK cells from patients with CHS had normal cytokine compartments and cytokine secretion. CONCLUSION: LYST is involved in regulation of multiple aspects of NK cell lytic activity, ranging from governance of lytic granule size to control of their polarization and exocytosis, as well as regulation of endolysosomal compartment identity. LYST functions in the regulated exocytosis but not in the constitutive secretion pathway.

Incidence and clinical presentation of primary hemophagocytic lymphohistiocytosis in Sweden.

BACKGROUND: Primary hemophagocytic lymphohistiocytosis (HLH) represents a group of inherited hyperinflammatory immunodeficiencies, including familial HLH (FHL), Griscelli syndrome type 2 (GS2), and X-linked lymphoproliferative syndrome (XLP). We previously reported an annual incidence of suspected primary HLH in Sweden 1971-1986 of 0.12 per 100,000 children. Here, we determined if the incidence had increased with concomitant awareness. PROCEDURE: Children <15 years old presenting with HLH 1987-2006 in Sweden were identified through the national mortality registry as well as by nation-wide inquiries to all pediatric centers. HLH was diagnosed according to the HLH-2004 diagnostic guidelines (in case of missing data of at least three of the eight diagnostic criteria, fulfillment of four was sufficient for inclusion). We defined primary HLH as patients presenting with HLH requiring transplantation or dying of disease. RESULTS: Remarkably, the minimal annual incidence rate of primary HLH remained 0.12 per 100,000 children, equating to 1.8 per 100,000 live births. Notably, an increased overall survival was observed in 1997-2006, relative to the period 1987-1996. During the subsequent 5-year period, 2007-2011, the incidence of genetically and/or functionally verified primary HLH was 0.15 per 100,000 children per year, suggesting that new assays may aid the identification of patients with primary HLH. CONCLUSION: The annual incidence of primary HLH in Sweden is 0.12-0.15 per 100,000 children per year. Pediatr Blood Cancer 2015;62:346-352. © 2014 Wiley Periodicals, Inc.

Transcriptomic profiling of progesterone in the male fathead minnow (Pimephales promelas) testis.

P4 is a hormone with diverse functions that include roles in reproduction, growth, and development. The objectives of this study were to examine the effects of P4 on androgen production in the mature teleost testis and to identify molecular signaling cascades regulated by P4 to improve understanding of its role in male reproduction. Fathead minnow (FHM) testis explants were treated in vitro with two concentrations of P4 (10(-8) and 10(-6) M) for 6 and 12 h. P4 significantly increased testosterone (T) production in the FHM testis but did not affect 11-ketotestosterone. Gene network analysis revealed that insulin growth factor (Igf1) and tumor necrosis factor receptor (Tnfr) signaling was significantly depressed with P4 treatment after 12h. There was also a 20% increase in a gene network for follicle-stimulating hormone secretion and an 18% decrease in genes involved in vasopressin signaling. Genes in steroid metabolism (e.g. star, cyp19a, 11bhsd) were not significantly affected by P4 treatments in this study, and it is hypothesized that pre-existing molecular machinery may be more involved in the increased production of T rather than the de novo expression of steroid-related transcripts and receptors. There was a significant decrease in prostaglandin E synthase 3b (cytosolic) (ptges3b) after treatment with P4, suggesting that there is cross talk between P4 and prostaglandin pathways in the reproductive testis. P4 has a role in regulating steroid production in the male testis and may do so by modulating gene networks related to endocrine pathways, such as Igf1, Tnfr, and vasopressin.

Activation of Cytotoxic Lymphocytes Through CD6 Enhances Killing of Cancer Cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8+ T cells. Tumor-infiltrating cytotoxic lymphocytes were found in higher proportions and were activated in UMCD6-treated mice compared to controls. Similar changes in gene expression were observed by RNA-seq analysis of NK cells treated with UMCD6. Particularly, UMCD6 up-regulated the NKG2D-DAP10 complex and activated PI3K. Thus, the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

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.

A Novel Tri-Functional Liposome Re-Educates "Cold Tumor" and Abrogates Tumor Growth by Synergizing Autophagy Inhibition and PD-L1 Blockade.

Immunotherapy has been regarded as a breakthrough in cancer treatment and achieved great success. However, the poor response rate is still a formidable challenge of current immunotherapies, especially in solid tumors without sufficient infiltration of immune cells, also known as "cold tumor." SAR405 is a highly specific VPS34 inhibitor and has been suggested as a potential approach converting "cold tumor" into "hot tumor" by inhibiting autophagy. In this study, a tri-functional doxorubicin (DOX) plus SAR405 liposome system is established and further modified with a novel anti-PD-L1 peptide JY4 for targeted delivery (DOX-SAR-JY4LIPO ). The data here demonstrate that in a lung cancer xenograft mouse model, by facilitating the tumoral enrichment of both SAR405 and DOX, DOX-SAR-JY4LIPO effectively increases the infiltration of cytotoxic lymphocytes in the tumor by synergizing DOX-induced immunogenic cell death (ICD) and SAR405-mediated upregulation of chemokines including CCL5 and CXCL10. As results, DOX-SAR-JY4LIPO significantly inhibits tumor growth, metastasis, and resurrection by re-educating immunosuppressive tumor microenvironment. In conclusion, this study not only proves the concept of inhibiting autophagy for better immune infiltration in the tumor but also presents a novel tri-functional liposomal system that overcomes the deficiencies of current therapies and holds great promise in cancer immunotherapy.