Stem cell-like memory T cells: A perspective from the dark side.

Much attention has been paid to a newly discovered subset of memory T (TM) cells-stem cell-like memory T (TSCM) cells for their high self-renewal ability, multi-differentiation potential and long-term effector function in adoptive therapy against tumors. Despite their application in cancer therapy, an excess of TSCM cells also contributes to the persistence of autoimmune diseases for their immune memory and HIV infection as a long-lived HIV reservoir. Signaling pathways Wnt, AMPK/mTOR and NF-κB are key determinants for TM cell generation, maintenance and proinflammatory effect. In this review, we focus on the phenotypic and functional characteristics of TSCM cells and discuss their role in autoimmune diseases and HIV-1 chronic infection. Also, we explore the potential mechanism and signaling pathways involved in immune memory and look into the future therapy strategies of targeting long-lived TM cells to suppress pathogenic immune memory.

Hypomorphic variants in AK2 reveal the contribution of mitochondrial function to B-cell activation.

BACKGROUND: The gene AK2 encodes the phosphotransferase adenylate kinase 2 (AK2). Human variants in AK2 cause reticular dysgenesis, a severe combined immunodeficiency with agranulocytosis, lymphopenia, and sensorineural deafness that requires hematopoietic stem cell transplantation for survival. OBJECTIVE: We investigated the mechanisms underlying recurrent sinopulmonary infections and hypogammaglobulinemia in 15 patients, ranging from 3 to 34 years of age, from 9 kindreds. Only 2 patients, both of whom had mildly impaired T-cell proliferation, each had a single clinically significant opportunistic infection. METHODS: Patient cells were studied with next-generation DNA sequencing, tandem mass spectrometry, and assays of lymphocyte and mitochondrial function. RESULTS: We identified 2 different homozygous variants in AK2. AK2G100S and AK2A182D permit residual protein expression, enzymatic activity, and normal numbers of neutrophils and lymphocytes. All but 1 patient had intact hearing. The patients' B cells had severely impaired proliferation and in vitro immunoglobulin secretion. With activation, the patients' B cells exhibited defective mitochondrial respiration and impaired regulation of mitochondrial membrane potential and quality. Although activated T cells from the patients with opportunistic infections demonstrated impaired mitochondrial function, the mitochondrial quality in T cells was preserved. Consistent with the capacity of activated T cells to utilize nonmitochondrial metabolism, these findings revealed a less strict cellular dependence of T-cell function on AK2 activity. Chemical inhibition of ATP synthesis in control T and B cells similarly demonstrated the greater dependency of B cells on mitochondrial function. CONCLUSIONS: Our patients demonstrate the in vivo sequelae of the cell-specific requirements for the functions of AK2 and mitochondria, particularly in B-cell activation and antibody production.

Leishmania major adenylate kinase immunization offers partial protection to a susceptible host.

Leishmania major causes mild-to-severe cutaneous lesions resulting in significant disfigurations, if untreated. The drugs are toxic, and drug-resistance parasites are emerging. Therefore, a prophylactic vaccination is an urgent need. As no vaccine is available, we compared the genes expressed by virulent and avirulent parasites. We identify L major adenylate kinase (AdeK) as a probable vaccine candidate after a series of experimentations. We cloned the gene in mammalian pcDNA6/HisA and pet28a+ vector for in vivo expression following immunization and in vitro protein expression for booster, respectively. We observed that immunization of susceptible BALB/c mice with AdeK resulted in significant protection against L major challenge infection. The protection was accompanied by increased IFN-γ producing lymphocytes and reduced IL-4, IL-17 and IL-10 secreting central and effector Th2, Th17 and Treg memory cells, respectively. These observations indicate L major AdeK as a potential vaccine candidate.

Isovitexin-Mediated Regulation of Microglial Polarization in Lipopolysaccharide-Induced Neuroinflammation via Activation of the CaMKKß/AMPK-PGC-1α Signaling Axis.

Microglia are the brain's immune cells and play an important role in regulating the microenvironment in the central nervous system. Activated microglia are capable of acquiring the pro-inflammatory (M1) phenotype and anti-inflammatory (M2) phenotype. Overactivation of microglia is neurotoxic and may lead to neuroinflammatory brain disorders. Neuroinflammation in the brain plays a crucial role part in the pathophysiology of many psychiatric and neurological diseases. The inhibition of M1 microglia and promotion of M2 microglia was demonstrated to treat and prevent these diseases through reduced neuroinflammation. Isovitexin (IVX) has anti-inflammatory properties and passes through the blood-brain barrier; however, the molecular mechanism that modulates IVX-mediated microglial polarization remains unclear. In BV-2 cells and mouse primary microglia, IVX suppressed the expression of M1 microglial markers, enhanced the expression of M2 microglial markers, and enhanced the release of interleukin 10 (IL-10). IVX promoted the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and PPARγ coactivator-1α (PGC-1α) in LPS-induced microglial activation. The inhibition of PPARγ and PGC-1α attenuated the regulatory effect of IVX in LPS-induced microglial polarization. IVX increased the expression of p-CaMKKß, p-AMPK, and PGC-1α in BV-2 cells. Inhibition of CaMKKß with STO-609 or knockdown of CaMKKß with CaMKKß siRNA attenuated IVX-mediated M2 microglial polarization in LPS-treated cells. In LPS-treated mice, the inhibition of CaMKKß and PGC-1α attenuated the IVX-mediated prevention of sickness behavior and enhanction of IVX-mediated M2 microglial polarization. IVX promoted M2 microglial polarization which exerted anti-inflammatory effects on LPS-induced neuroinflammation via the activation of the CaMKKß/AMPK-PGC-1α signaling axis.

TLR-2 and MyD88-Dependent Activation of MAPK and STAT Proteins Regulates Proinflammatory Cytokine Response and Immunity to Experimental Trypanosoma congolense Infection.

It is known that Trypanosoma congolense infection in mice is associated with increased production of proinflammatory cytokines by macrophages and monocytes. However, the intracellular signaling pathways leading to the production of these cytokines still remain unknown. In this paper, we have investigated the innate receptors and intracellular signaling pathways that are associated with T. congolense-induced proinflammatory cytokine production in macrophages. We show that the production of IL-6, IL-12, and TNF-α by macrophages in vitro and in vivo following interaction with T. congolense is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) including ERK, p38, JNK, and signal transducer and activation of transcription (STAT) proteins. Specific inhibition of MAPKs and STATs signaling pathways significantly inhibited T. congolense-induced production of proinflammatory cytokines in macrophages. We further show that T. congolense-induced proinflammatory cytokine production in macrophages is mediated via Toll-like receptor 2 (TLR2) and involves the adaptor molecule, MyD88. Deficiency of MyD88 and TLR2 leads to impaired cytokine production by macrophages in vitro and acute death of T. congolense-infected relatively resistant mice. Collectively, our results provide insight into T. congolense-induced activation of the immune system that leads to the production of proinflammatory cytokines and resistance to the infection.

Identification of adenylate kinase 5 antibodies during routine diagnostics in a tissue-based assay: Three new cases and a review of the literature.

Antibodies against adenylate kinase 5 (AK5) have been described in patients with non-paraneoplastic limbic encephalitis, mainly in men around 70 years of age. Routine testing with specific cell-based assays is not yet available. Three patients with episodic anterograde memory problems and depression had extensive limbic lesions and developed severe atrophy, mainly of the medial temporal lobes. The antibodies were identified in serum and CSF based on the typical staining pattern of AK5 antibodies on a tissue-based assay (here, unfixed mouse brain). Subsequently, they were confirmed by a research laboratory through a cell-based assay.

Recombinant adenylate kinase 3 from liver fluke Clonorchis sinensis for histochemical analysis and serodiagnosis of clonorchiasis.

Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

Adipose Tissue-Specialized Immunologic Features Might Be the Potential Therapeutic Target of Prospective Medicines for Obesity.

Excessive lipid accumulation in adipose tissue is either the source of obesity or the cause and result of chronic local inflammation, and recent studies indicate that the accumulation may induce many other specialized immunologic features with macrophages and epidemic diseases. We analyze the effective stages of immune cells in adipose tissue, including macrophage recruitment, macrophage polarization, and macrophage-like phenotype preadipocyte possession to find optimal sites as drug targets. Subsequently, some main signaling pathways are summarized in this review, including the AMP-activated protein kinase (AMPK) pathway, the JNK signaling pathway, and a novel one, the Notch signaling pathway. We illustrate all these points in order to determine the general pathogenesis of chronic low-grade local inflammation in adipose tissue and the related signaling pathways. In addition, signal-associated prospective compounds, such as berberine, are summarized and discussed with potential targets in pathogenesis. This might provide some possible thoughts and novel therapies for studying chronic inflammatory diseases, such as insulin resistance and type 2 diabetes mellitus.

Immunization with recombinant schistosome adenylate kinase 1 partially protects mice against Schistosoma japonicum infection.

Highly effective and safe prophylactic vaccines are urgently needed to sustainably control schistosomiasis, one of the most serious endemic zoonoses in China. In this study, we characterized adenylate kinase 1 from Schistosoma japonicum (SjAK1), a phosphotransferase that regulates cellular energy and metabolism, and evaluated its potential as a recombinant vaccine. Based on real-time quantitative PCR, western blot, and immunolocalization, SjAK1 is active throughout the life of the worm, although its expression is higher in 21-day-old schistosomula, adult worms, and eggs deposited in the host liver. Further, the enzyme accumulates in the eggshell, intestinal epithelium, integument of adult worms and in the vitellaria tissue in female worms. A 594-bp full-length complementary DNA (cDNA) encoding SjAK1 was synthesized from total RNA of 3-day-old schistosomes, and immunization with recombinant SjAK1 reduced worm burden by 50%, decreased the density of eggs deposited in the host liver by 40%, and reduced the area of granulomas in the host liver by 56%. ELISA results showed that recombinant SjAK1 also stimulated Th1 cytokines such as IL-2 and IFN-γ, but not IL-5 and IL-4. Collectively, a recombinant form of the enzyme SjAK1 elicits partial protective immunity against Schistosoma japonicum infection and the induction of Th1 cytokines. Thus, the enzyme has potential as a component of a multivalent vaccine against schistosomiasis.

Characteristics in limbic encephalitis with anti-adenylate kinase 5 autoantibodies.

OBJECTIVE: To report 10 patients with limbic encephalitis (LE) and adenylate kinase 5 autoantibodies (AK5-Abs). METHODS: We conducted a retrospective study in a cohort of 50 patients with LE with uncharacterized autoantibodies and identified a specific target using immunohistochemistry, Western blotting, immunoprecipitation, mass spectrometry, and cell-based assay. RESULTS: AK5 (a known autoantigen of LE) was identified as the target of antibodies in the CSFs and sera of 10 patients with LE (median age 64 years; range 57-80), which was characterized by subacute anterograde amnesia without seizure and sometimes preceded by a prodromal phase of asthenia or mood disturbances. Anterograde amnesia can be isolated, but some patients also complained of prosopagnosia, paroxysmal anxiety, or abnormal behavior. No associated cancer was observed. All 10 patients had bilateral hippocampal hypersignal on a brain MRI. CSF analysis generally showed a mild pleiocytosis with elevated immunoglobulin G index and oligoclonal bands, as well as high levels of tau protein with normal concentration of Aß42 and phospho-tau, suggesting a process of neuronal death. Except for one patient, clinical response to immunotherapy was unfavorable, with persistence of severe anterograde amnesia. Two patients evolved to severe cognitive decline. Hippocampal atrophy was observed on control brain MRI. Using in vitro tests on hippocampal neurons, we did not identify clues suggesting a direct pathogenic role of AK5-Abs. CONCLUSIONS: AK5-Abs should be systematically considered in aged patients with subacute anterograde amnesia. Recognition of this disorder is important to develop new treatment strategies to prevent irreversible limbic damage.

AMP kinase promotes Bcl6 expression in both mouse and human T cells.

The transcription factor Bcl6 is a master regulator of follicular helper T (TFH) cells, and understanding the signaling pathway that induces Bcl6 and TFH cell differentiation is therefore critical. IL-2 produced during T cell activation inhibits Bcl6 expression but how TFH cells evade IL-2 inhibition is not completely understood. Here we show that Bcl6 is highly up-regulated in activated CD4 T cells following glucose deprivation (GD), and this pathway is insensitive to inhibition by IL-2. Similar to GD, the glucose analog 2-deoxyglucose (2DG) inhibits glycolysis, and 2DG induced Bcl6 expression in activated CD4 T cells. The metabolic sensor AMP kinase (AMPK) is activated when glycolysis is decreased, and the induction of Bcl6 by GD was inhibited by the AMPK antagonist compound C. Additionally, activation of AMPK by the drug AICAR caused Bcl6 up-regulation in activated CD4 T cells. When mice were immunized with KLH using AICAR as an adjuvant, there was a strong TFH-dependent enhancement of KLH-specific antibody (Ab) responses, and higher Bcl6 expression in TFH cells in vivo. Activation of AMPK strongly induced BCL6 and the up-regulation of TFH cell marker expression by human CD4 T cells. Our data reveal a major new pathway for TFH cell differentiation, conserved by both mouse and human T cells. Mature TFH cells are reported to have a lower metabolic state compared to TH1 cells. Our data indicates that decreased metabolism may be deterministic for TFH cell differentiation, and not simply a result of TFH cell differentiation.

Adenylate kinase: a novel antigen for immunodiagnosis and subunit vaccine against tuberculosis.

UNLABELLED: Mycobacterium tuberculosis (M.tb)-derived antigens capable of inducing strong cellular and/or humoral responses are potential targets for both immunodiagnosis and vaccine development against tuberculosis (TB). In the present study, we identified adenylate kinase (ADK, Rv0733) as an antigen that induces high cellular and antibody responses in active TB patients. We consequently tested the use of ADK-specific T cells and antibodies as biomarkers for TB diagnosis. The ADK-specific IFN-γ-producing cells detected by ELISPOT assay showed a sensitivity of 85.0 % and specificity of 94.15 % for TB diagnosis while ADK-specific IgG antibody showed a sensitivity of 40.35 % and specificity of 96.43 %. Combining ADK-specific cellular and antibody responses increased the sensitivity to 91.59 % and the specificity to 96.15 %. Immunogenicity and protection against M.tb infection were further tested in a murine model. Immunization with ADK protein elicited strong specific T- and B-cell responses, and provided protection against the virulent H37Rv stain of M.tb resulting in lower bacilli load in the spleens and lungs. More ADK-specific polyfunctional Th1 cells were observed in the lungs when compared to adjuvant-immunized mice. ADK thus may serve as a novel M.tb antigen for TB immunodiagnosis and development of subunit vaccines. KEY MESSAGES: ADK induces strong immune responses both in humans and mice. ADK-specific IFN-γ production and B-cell responses have high potential for TB diagnosis. ADK immunization provides protection against M.tb infection.

Clinico-pathological correlation in adenylate kinase 5 autoimmune limbic encephalitis.

Autoantibodies associated with autoimmune limbic encephalitis (ALE) have been well-characterized, with intracellular neuronal antibodies being less responsive to immunotherapy than antibodies to cell surface antigens. Adenylate kinase 5 (AK5) is a nucleoside monophosphate kinase vital for neuronal-specific metabolism and is located intracellularly in the cytosol and expressed exclusively in the brain. Antibodies to AK5 had been previously identified but were not known to be associated with human disease prior to the report of two patients with AK5-related ALE (Tuzun et al., 2007). We present the complete clinical picture for one of these patients and the first reported neuropathology for AK5 ALE.

Inborn errors of the development of human natural killer cells.

PURPOSE OF REVIEW: Inborn errors of human natural killer (NK) cells may affect the development of these cells, their function, or both. There are two broad categories of genetic defects of NK cell development, depending on whether the deficiency is apparently specific to NK cells or clearly affects multiple hematopoietic lineages. We review here recent progress in the genetic dissection of these NK deficiencies (NKDs). RECENT FINDINGS: Patients with severe combined immunodeficiencies bearing mutations of adenosine deaminase, adenylate kinase 2, interleukin-2 receptor gamma chain, and Janus kinase 3 genes present NKDs and are prone to a broad range of infections. Patients with GATA binding protein 2 deficiency are susceptible to both mycobacterial and viral infections, and display NKDs and a lack of monocytes. Rare patients with mini chromosomal maintenance 4 deficiency display an apparently selective NKD associated with viral infections, but they also display various nonhematopoietic phenotypes, including adrenal insufficiency and growth retardation. SUMMARY: These studies have initiated a genetic dissection of the development of human NK cells. Further studies are warranted, including the search for genetic causes of NKD in particular. This research may lead to the discovery of molecules specifically controlling the development of NK cells and to improvements in our understanding of the hitherto elusive function of these cells in humans.

The chemokine CCL5 regulates glucose uptake and AMP kinase signaling in activated T cells to facilitate chemotaxis.

Recruitment of effector T cells to sites of infection or inflammation is essential for an effective adaptive immune response. The chemokine CCL5 (RANTES) activates its cognate receptor, CCR5, to initiate cellular functions, including chemotaxis. In earlier studies, we reported that CCL5-induced CCR5 signaling activates the mTOR/4E-BP1 pathway to directly modulate mRNA translation. Specifically, CCL5-mediated mTOR activation contributes to T cell chemotaxis by initiating the synthesis of chemotaxis-related proteins. Up-regulation of chemotaxis-related proteins may prime T cells for efficient migration. It is now clear that mTOR is also a central regulator of nutrient sensing and glycolysis. Herein we describe a role for CCL5-mediated glucose uptake and ATP accumulation to meet the energy demands of chemotaxis in activated T cells. We provide evidence that CCL5 is able to induce glucose uptake in an mTOR-dependent manner. CCL5 treatment of ex vivo activated human CD3(+) T cells also induced the activation of the nutrient-sensing kinase AMPK and downstream substrates ACC-1, PFKFB-2, and GSK-3ß. Using 2-deoxy-d-glucose, an inhibitor of glucose uptake, and compound C, an inhibitor of AMPK, experimental data are presented that demonstrate that CCL5-mediated T cell chemotaxis is dependent on glucose, as these inhibitors inhibit CCL5-mediated chemotaxis in a dose-dependent manner. Altogether, these findings suggest that both glycolysis and AMPK signaling are required for efficient T cell migration in response to CCL5. These studies extend the role of CCL5 mediated CCR5 signaling beyond lymphocyte chemotaxis and demonstrate a role for chemokines in promoting glucose uptake and ATP production to match energy demands of migration.

Combined cryoablation and GM-CSF treatment for metastatic hormone refractory prostate cancer.

A total of 12 patients with metastatic hormone refractory prostate cancer were treated by combining cryoablation and granulocyte macrophage colony-stimulating factor administration. Besides prostate-specific antigen (PSA) measurements, peripheral blood mononuclear cells were also obtained; the frequency of tumor-specific T cells was tested ex vivo in an interferon-gamma enzyme-linked immunospot assay after stimulating with autologous prostate cancer-derived protein lysates. To assess cytolytic activity, T cells were coincubated with human prostate cancer cells (LNCaP) or renal cancer cells (GRC-1), and release of cytosolic adenylate kinase was measured by a luciferase assay. The median PSA decline percentage was 69.4% (range: 30.5% to 92.5%) and the median time to the nadir PSA was 4 months after therapy (range: 3 to 6). The median time to disease progress was 18 months, and 1 patient obtained a 92.5% PSA decline and a greater than 50% reduction of lung disease and survived 31 months. Four or 8 weeks after treatment, the tumor-specific T-cell responses were increased in peripheral blood mononuclear cell. The cytolytic activity against LNCaP was also increased significantly whereas no response was found against GRC-1. It seemed that there was no direct correlation between the degree of T-cell response and decline in PSA. Combined cryoablation with granulocyte macrophage colony-stimulating factor treatment was suggested to be an alternative approach for metastatic hormone refractory prostate cancer and could induce tumor-specific immunologic response.

Adenylate kinase 5 autoimmunity in treatment refractory limbic encephalitis.

We report two men with limbic encephalitis (LE) refractory to corticosteroids, IVIg and plasma exchange. Both patients had serum/CSF antibodies that reacted with the cytoplasm of neurons. Probing of a hippocampal cDNA library resulted in the isolation of adenylate kinase 5 (AK5). Patients' antibodies, but not those of 111 controls, recognized AK5-expressing phage plaques. Human AK5-affinity purified antibodies reproduced the neuronal immunolabeling of patients' antibodies, and co-localized with a rabbit AK5 antibody, confirming that the brain autoantigen was AK5. Detection of antibodies to AK5 in LE patients carries a poor prognosis, and suggests the prompt use of aggressive immunosuppression.

[Expression of adenylate kinase of Schistosoma japonicum and evaluation on the immunoreactivity of the recombinant protein].

OBJECTIVE: To express and purify the Schistosoma japonicum adenylate kinase (AK) protein of which the cDNA sequence was subcloned into the pET32a(+) soluble expression plasmid, and to evaluate its immunoreactivity. METHODS: The recombinant plasmid was transformed into E. coli BL21, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS-PAGE after boiling and centrifugation. The target protein was purified with the Ni-NTA agarose. The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti-6-His antibody, sera from rabbits 6 weeks after infected with cercariae or UV-attenuated cercariae. The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum. RESULTS: The molecular weight of the target fusion protein was Mr 40 000 after being induced with IPTG. The fused protein showed a single band when reacted with anti-6-His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum. CONCLUSION: The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about Mr 40000. This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV-attenuated cercariae.