T-lymphocyte Production Research Articles

In silico assessment of CAR macrophages activity against SARS-CoV-2 infection

Macrophage engineering with chimeric antigen receptor is a promising technique first applied to the treatment of tumours and recently suggested as a possible immunotherapeutic route against the COVID-19 disease. Four immunotherapies based on engineered macrophages have been tested in vitro revealing promising, with one of them acting without increasing the cytokines level. We present a mathematical model aimed at the evaluation of both the SARS-CoV-2 virions dynamics and the cytokines production induced, while such newly developed constructs interact with the immune system once administered. The importance of the study lies both in monitoring the dynamics of the infection and in evaluating the cytokine production, since clinical studies show that in critical COVID-19 patients an abnormal cytokines production occurs, a concern to be accounted for in designing appropriate therapeutic strategies. The mathematical model was built in the context of the continuum approach of the mass conservation, while the numerical simulations have been performed introducing parameters deduced from the experiments, using the finite element method. The model simulations allow to analyse and to compare the immune mechanisms underlying the virus dynamics, deepening the investigation for two selected immunotherapies, suggesting that a synergistic work of involved cytokines with phagocytic activity of macrophages occurs. The best SARS-CoV-2 clearance relies not only on the phagocytic capacity of the engineered macrophages, but also on the production of T-lymphocytes, pro- and anti-inflammatory cytokines which in the two cases examined in depth can decrease by 99.7 %, 99.6 % and 69 % respectively, passing from the most effective immunotherapy to the least effective one. This study is the first mathematical model that analyses the dynamics of macrophages engineered to fight the COVID-19, and paves the way for their possible exploitation against such a challenging disease, going beyond existing models involving other immune cells.

Patient-tailored adoptive immunotherapy with EBV-specific T cells from related and unrelated donors.

BACKGROUNDAdoptive transfer of EBV-specific T cells can restore specific immunity in immunocompromised patients with EBV-associated complications.METHODSWe provide results of a personalized T cell manufacturing program evaluating donor, patient, T cell product, and outcome data. Patient-tailored clinical-grade EBV-specific cytotoxic T lymphocyte (EBV-CTL) products from stem cell donors (SCDs), related third-party donors (TPDs), or unrelated TPDs from the allogeneic T cell donor registry (alloCELL) at Hannover Medical School were manufactured by immunomagnetic selection using a CliniMACS Plus or Prodigy device and the EBV PepTivators EBNA-1 and Select. Consecutive manufacturing processes were evaluated, and patient outcome and side effects were retrieved by retrospective chart analysis.RESULTSForty clinical-grade EBV-CTL products from SCDs, related TPDs, or unrelated TPDs were generated for 37 patients with refractory EBV infections or EBV-associated malignancies with and without a history of transplantation, within 5 days (median) after donor identification. Thirty-four patients received 1-14 EBV-CTL products (fresh and cryopreserved). EBV-CTL transfer led to a complete response in 20 of 29 patients who were evaluated for clinical response. No infusion-related toxicity was reported. EBV-specific T cells in patients' blood were detectable in 16 of 18 monitored patients (89%) after transfer, and their presence correlated with clinical response.CONCLUSIONPersonalized clinical-grade manufacture of EBV-CTL products via immunomagnetic selection from SCDs, related TPDs, or unrelated TPDs in a timely manner is feasible. Overall, EBV-CTLs were clinically effective and well tolerated. Our data suggest EBV-CTL transfer as a promising therapeutic approach for immunocompromised patients with refractory EBV-associated diseases beyond HSCT, as well as patients with preexisting organ dysfunction.TRIAL REGISTRATIONNot applicable.FUNDINGThis study was funded in part by the German Research Foundation (DFG, 158989968/SFB 900), the Deutsche Kinderkrebsstiftung (DKS 2013.09), Wilhelm-Sander-Stiftung (reference 2015.097.1), Ellen-Schmidt-Program of Hannover Medical School, and German Federal Ministry of Education and Research (reference 01EO0802).

Metabolic engineering in food crops to enhance ascorbic acid production: crop biofortification perspectives for human health.

Ascorbic acid (AsA) also known as vitamin C is considered as an essential micronutrient in the diet of humans. The human body is unable to synthesize AsA, thus solely dependent on exogenous sources to accomplish the nutritional requirement. AsA plays a crucial role in different physiological aspects of human health like bone formation, iron absorption, maintenance and development of connective tissues, conversion of cholesterol to bile acid and production of serotonin. It carries antioxidant properties and is involved in curing various clinical disorders such as scurvy, viral infection, neurodegenerative diseases, cardiovascular diseases, anemia, and diabetes. It also plays a significant role in COVID-19 prevention and recovery by improving the oxygen index and enhancing the production of natural killer cells and T-lymphocytes. In plants, AsA plays important role in floral induction, seed germination, senescence, ROS regulation and photosynthesis. AsA is an essential counterpart of the antioxidant system and helps to defend the plants against abiotic and biotic stresses. Surprisingly, the deficiencies of AsA are spreading in both developed and developing countries. The amount of AsA in the major food crops such as wheat, rice, maize, and other raw natural plant foods is inadequate to fulfill its dietary requirements. Hence, the biofortification of AsA in staple crops would be feasible and cost-effective means of delivering AsA to populations that may have limited access to diverse diets and other interventions. In this review, we endeavor to provide information on the role of AsA in plants and human health, and also perused various biotechnological and agronomical approaches for elevating AsA content in food crops.

Assessment of macrocyclic lacton group products for ectoparasitosis of carp

The material for the study were Lubin carp of one year old spontaneously invaded by the ectoparasites Lernaea cyprinacea and Dactylogyrus vastator. For the experiment we used drugs: "Brovermectin-granulate" (development of “BFP” serial production; 1 g of the preparation contains: active substance (ADR) ivermectin – 3.5 mg; tocopherol acetate (20 mg) and Emamectin benzoate (manufactured by King Quenson Industry Group; 1 g of preparation contains ADR emamectin benzoate (50 mg). Analyzing morphological, biochemical and immunological parameters of blood and organs of fish, it is possible to state that the fact that the anti-parasitic drugs “Brovermectin granulate” and “Emamectin benzoate” has a pronounced antiparasitic effect, normalizing the homeostasis of the body. The results obtained showed significant fluctuations in the number neutrophils, eosinophils and lymphocytes. In ectoparasites infested of fishes used for macrocyclic lactones, y leukocyte formula sharply increases the percentage of rod-shaped neutrophils and at the same time decreases the proportion of lymphocytes. At the same time, there is an increase in the number of eosinophils that perform the function of protecting the body of fish from parasites. Eosinophilia, as in the higher vertebrates, is one of objective indicators of allergy (sensitization) of the body, nature the course of inflammatory processes. The results obtained indicate a decrease in the production of total T-lymphocytes are cells that play a key role in the immune system protection in the body of carp for damage by ectoparasites. In analyzing the above data, attention is drawn to the lower functional the activity of the blood T-lymphocyte system in ectoparasites infested with fish, than those given antiparasitic drugs. From this it follows that the ability of the blood lymphocytes of the fish to which the drugs from the group were given of macrocyclic lactones, before pathogen binding and production of antigens the antibodies that neutralize them are much higher than the fish affected ectoparasites. The immune system in fish provides self-regulation through direct contact of cells (macrophages, neutrophils, cytotoxic T-lymphocytes) as well as due to humoral factors (lysozyme, complement). The use of drugs from the group of macrocyclic lactones promotes the release of infested fish from ectoparasites with the following normalization of their life, increase of immune status and resistance.

The bidirectional interaction between the sympathetic nervous system and immune mechanisms in the pathogenesis of hypertension.

Over the last few years, evidence has accumulated to suggest that hypertension is, at least in part, an immune-mediated inflammatory disorder. Many links between immunity and hypertension have been established and provide a complex framework of mechanistic interactions contributing to the rise in BP. These include immune-mediated inflammatory processes affecting regulatory brain nuclei and interactions with other mediators of cardiovascular regulation such as the sympathetic nervous system. Sympathoexcitation differentially regulates T-cells based upon activation status of the immune cell as well as the resident organ. Exogenous and endogenous triggers activate signalling pathways in innate and adaptive immune cells resulting in pro-inflammatory cytokine production and activation of T-lymphocytes in the cardiovascular and renal regions, now considered major factors in the development of essential hypertension. The inflammatory cascade is sustained and exacerbated by the immune flow via the brain-bone marrow-spleen-gastrointestinal axis and thereby further aggravating immune-mediated pathways resulting in a vicious cycle of established hypertension and target organ damage. This review summarizes the evidence and recent advances in linking immune-mediated inflammation, sympathetic activation and their bidirectional interactions with the development of hypertension. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

Evolution of the views on pathogenesis of autoimmune thyroid diseases and
 prospects for their target therapy

Modern scientific literature contains few reports concerning the influence of target therapy on pathogenetic factors of autoimmune thyroid diseases (AITD). Despite a large number of hypotheses of AITD pathogenesis, the only well established fact is the starting stage of Graves disease (GD) and autoimmune thyroiditis (AIT) is the loss of tolerance to thyroid autoantigens and the final stage is production of autoantibodies to them. Up to 75-80% of the patients with GD have antibodies against thyroid peroxidase and only few of them have anti-thyroglobulin antibodies more characteristic of AIT. Thyrotropin releasing hormone (TRH) is known to stimulate T-lymphocyte production via local effect on insulin-like growth factor (IGF). Modern studies confirm the important role of cytokines in immunopathogenesis of GD and AIT. Moreover, excess activation of this system in AITD provides a basis for the development of specific therapeutic approaches to personified pharmacotherapy. The effectiveness of anti-cytokine therapy of GD and AIT was demonstrated in animal experiments. Studies of therapy targeted on orbital and thyroid autoantigens in AITD are currently underway. The existence of specific receptors and the ability of immunocompetent cells to produce neuropeptides create prerequisites for their participation in intercellular cooperative processes. It can be supposed, by analogy with the influence of hormones and neuromediators on immunocytes, that neurohormones act on them via specific receptors with the involvement of cyclic nucleotides. It opens up opportunity for targeted correction of these relationships. Further studies of immunopathogenetic mechanisms of GD and AIT for better understanding the role of interaction between inborn and acquired immunity, its regulation, and intersystem transmission of signals in the development of these diseases are needed to realize modern strategies of their target therapy.

Staphylococcus enterotoxin profile of China isolates and the superantigenicity of some novel enterotoxins.

The genus of staphylococcus widely distributes in environments and contributes to a variety of animal and human diseases. The enterotoxins (SEs) secreted by this type of pathogen have been the leading cause of bacterial toxic shock syndrome and food poisoning, and thus present a substantial concern to public health. In this study, we analyzed the superantigen profile of 122 staphylococcus strains isolated from diverse sources. When screened for the presence and prevalence of 17 known se or se-like (sel) genes, except selj, all other genes were detected in these isolates. In particular, 95.9% of the isolates harbored at least one se/sel gene. Moreover, 47.5% of them bore at least 5. Remarkably, several non-pathogenic species of animal- and environment-origin were also found to carry multiple se/sels. The most frequent genes detected were tsst (62.3%), sei (54.1%), and seb (46.7%), followed by some sel genes (selo, selu, and selm), which also were present at relatively high frequency (20-30%). The generated data improved understanding of strain-specific differences in enterotoxin expression. The gene products of the latter (selo and selu) were subsequently analyzed for their antigenicity in a mouse model using purified E. coli-based recombinant proteins. The studies revealed a strong activity for SEO in induction of T-lymphocyte proliferation and production of various inflammatory cytokines either in vivo or in vitro. In contrast, SEU exhibited little superantigenic effects. The molecular basis for the difference in antigenicity was analyzed by 3D homology remodeling, which revealed a difference in binding and affinities for MHC-II molecules and TCR Vβ region.

Vitamins: Key Role Players in Boosting Up Immune Response-A Mini Review

Vitamins perform different functions in our body and to boost up immune response towards pathogens is one of them. Immunity provides protection to life by its three main such as skin, cellular response, and humoral immune response. Both vitamins quality and quantity within the body promote systematic immune processes by regulating Tlymphocytes, antibodies, and cytokines formation. In this review, we will summarize the role of water soluble and fat soluble vitamins to boost up immunity functions. Vitamins A, C, and E mainly aid in enhancing the skin epithelium barrier function. With the exception of vitamin C, all the vitamins are claimed to be essential for antibody production. Most of the vitamins are applied in our body to produce a cell-mediated response with the production of cytokines and T-lymphocytes. Supplementation of the diet with vitamins in specifically selected appropriate quantities routinely can support body’s natural defence mechanism by enhancing the immune response.

Multisystem Anomalies in Severe Combined Immunodeficiency with Mutant BCL11B

BackgroundSevere combined immunodeficiency (SCID) is characterized by arrested T-lymphocyte production and by B-lymphocyte dysfunction, which result in life-threatening infections. Early diagnosis of SCID through population-based screening of newborns can aid clinical management and help improve outcomes; it also permits the identification of previously unknown factors that are essential for lymphocyte development in humans.MethodsSCID was detected in a newborn before the onset of infections by means of screening of T-cell–receptor excision circles, a biomarker for thymic output. On confirmation of the condition, the affected infant was treated with allogeneic hematopoietic stem-cell transplantation. Exome sequencing in the patient and parents was followed by functional analysis of a prioritized candidate gene with the use of human hematopoietic stem cells and zebrafish embryos.ResultsThe infant had “leaky” SCID (i.e., a form of SCID in which a minimal degree of immune function is preserved), as well as craniofacial and dermal abnormalities and the absence of a corpus callosum; his immune deficit was fully corrected by hematopoietic stem-cell transplantation. Exome sequencing revealed a heterozygous de novo missense mutation, p.N441K, in BCL11B. The resulting BCL11B protein had dominant negative activity, which abrogated the ability of wild-type BCL11B to bind DNA, thereby arresting development of the T-cell lineage and disrupting hematopoietic stem-cell migration; this revealed a previously unknown function of BCL11B. The patient’s abnormalities, when recapitulated in bcl11ba-deficient zebrafish, were reversed by ectopic expression of functionally intact human BCL11B but not mutant human BCL11B.ConclusionsNewborn screening facilitated the identification and treatment of a previously unknown cause of human SCID. Coupling exome sequencing with an evaluation of candidate genes in human hematopoietic stem cells and in zebrafish revealed that a constitutional BCL11B mutation caused human multisystem anomalies with SCID and also revealed a prethymic role for BCL11B in hematopoietic progenitors. (Funded by the National Institutes of Health and others.)

The inhibition of dopamine synthesis in fetuses changes the pattern of T-lymphocyte maturation in the thymus of adult rats.

The mRNA for dopamine receptors of type D1, D3, D5, but not type D2, was detected in the thymus of rats starting from day 16 of embryonic development (E16). Dopamine at concentrations of 10-8-10‒6 M inhibited fetus thymocyte response to mitogen, confirming the functionality of the receptors and the possibility of a direct effect of dopamine on the developing thymus. Pharmacological inhibition of catecholamine synthesis in the crucial period of thymus development leads to long-term changes in the T-system immunity due to increased production of natural regulatory T-lymphocytes. The presence and functional activity of dopamine receptors in the fetal thymus indicates its ability to influence the development of the immune system of rats during ontogeny.

685. Restoration of Antiviral Immunity Following Hematopoietic Stem Cell Transplantation with Virus-Specific T-Lymphocyte Therapy

Background: Adoptive immunotherapy using virus-specific cytotoxic T-lymphocyte (VST) products has been successful in restoring antiviral immunity after hematopoietic stem cell transplantation (HSCT). VST may be produced from a stem cell donor in 10-14 days using current rapid protocols, or banked, partially-HLA matched VST may be used as an “off the shelf” treatment. Objective: To evaluate the clinical efficacy of HSCT donor-derived VST and third-party VST for prevention or treatment of CMV, EBV, and adenovirus following HSCT. Design/Methods: VST were cultured from HSCT or third-party donors using a GMP-compliant rapid-manufacture protocol. VST were tested for specificity and non-alloreactivity via IFN-γ ELISpot and cytotoxicity assays. Patients were monitored for 45 days following infusion for toxicity and for up to 22 months for immune reconstitution. Results: Nineteen patients received VST on existing protocols, of which 15 products were derived from HSCT donors, and 6 were from partially matched, third-party donors. Sixteen patients were treated for CMV (N=11), EBV (N=5), and/or adenovirus infections(n=3), of which fourteen (88%) had partial or complete antiviral responses. Immune reconstitution against targeted viruses was seen using IFN-g ELISpot at a median time of 28 days following infusion. Three patients developed grade I-II GVHD within 45 days of VST infusion, all of which was treatment responsive. Sixteen of the 19 patients remain alive and free of active viral infection up to 22 months post-VST infusion. Conclusions: VST are effective for the restoration of antiviral following HSCT. Expansion of targeted viruses may further extend the utility of this therapy.

HIV Progression Perturbs the Balance of the Cell-Mediated and Anti-Inflammatory Adaptive and Innate Mycobacterial Immune Response.

Introduction. Our objective is to understand how HIV infection increases the risk of progression from latent tuberculosis (TB) to active disease. We understand now that immunity is a balance of competing immune responses by multiple cell types. Since T-lymphocyte production of interferon-gamma (IFN-γ) in response to Mycobacterium tuberculosis (Mtb) antigens fails to differentiate disease from latent infection, we applied a comprehensive profiling methodology to define immune biomarkers that reliably predict a patient's TB risk. Methods. We established a cohort of HIV-infected adults with TB disease from Swaziland. Multiparametric flow cytometry was used to quantify the mycobacterial-specific anti-inflammatory (IL-4 and IL-10) and proinflammatory (IFN-γ) immune response. Results. From 12 HIV-infected Swaziland patients with TB disease, the CD4+, CD8+, Double Negative, and CD56+CD3− lymphocytes increase their IL-4 : IFN-γ ratio as HIV disease worsens (Spearman r of −0.59; −0.59; −0.60; and −0.59, resp.; p < 0.05). Similarly, HIV severity is associated with an increased IL-10 : IFN-γ ratio (Spearman r of −0.76; p = 0.01). Conclusion. As HIV disease progresses, both the adaptive and innate branches skew away from an inflammatory and towards anti-inflammatory phenotype.

FORMATION OF INNATE AND ADAPTIVE IMMUNE RESPONSE UNDER THE INFLUENCE OF DIFFERENT FLAVIVIRUS VACCINES

The review examines in a comparative perspective the key moments of formation of innate and adaptive immune responses to different types of current flavivirus vaccines: live attenuated against yellow fever virus and inactivated whole virus against tick-borne encephalitis virus. Particular attention is paid to the ability of these different vaccines, containing exogenous pathogen-associated molecular structures, to stimulate innate immunity. Live attenuated vaccine by infecting several subtypes of dendritic cells activates them through various pattern-recognition receptors, such as Tolland RIG-I-like receptors, which leads to significant production of proinflammatory cytokines, including interferon-α primary mediator of innate antiviral immunity. By simulating natural viral infection, this vaccine quickly spreads over the vascular network, and the dendritic cells, activated by it, migrate to the draining lymph nodes and trigger multiple foci of Tand B-cell activation. Inactivated vaccine stimulates the innate immunity predominantly at the injection site, and for the sufficient activation requires the presence in its composition of an adjuvant (aluminum hydroxide), which effects the formation and activation of inflammasomes, ensuring the formation and secretion of IL-1β and IL-18 that, in turn, trigger a cascade of cellular and humoral innate immune responses. We demonstrated the possibility of involvement in the induction of innate immunity, mediated by the inactivated vaccine, endogenous pathogenassociated molecular patterns (uric acid and host cell DNA), forming at the vaccine injection site. We discuss the triggering of Band T-cell responses by flavivirus vaccines that determine various duration of protection against various pathogens. A single injection of the live vaccine against yellow fever virus induces polyvalent adaptive immune response, including the production of cytotoxic T-lymphocytes, Th1and Th2-cells and neutralizing antibodies, which may persist for up to 40 years after the vaccination. To induce and maintain protective immunity, mediated by the inactivated vaccine against tick-borne encephalitis virus, it is required: triple immunization, which results in the production primarily of neutralizing antibodies, and subsequent booster injections every 3 years. We considered the potential use of the data on immunological mechanisms of action of current vaccines to generate new highly efficient vaccines.

QS-21: A Potent Vaccine Adjuvant

QS-21 is one of the active fractions of the bark of Chilean tree, Quillaja saponaria, purified using a reverse-phase chromatography (RP-HPLC) [1–3]. QS denotes its source as Q. saponaria and the number 21 as the identity of the RP-HPLC peak [2]. QS-21 is an acylated 3, 28-bisdesmodic triterpene glycosides (1,3) or “saponin” with a molecular formula of C92O46H148 and molecular weight of 1990 Da [1]. It is one of the most potent immunological adjuvants that has been widely used [4–8]. The adjuvant effect of saponins was first reported in 1925, when it was shown that the addition of bread crumbs, tapioca, saponin and “starch oil” to antigenic preparations greatly enhanced antibody responses to diphtheria or tetanus [7,9–11]. In 1951, Espinet used a crude commercially available saponin preparation to increase the potency of foot-and-mouth disease vaccines [10,12]. Further in 1974, Dalsgaard successfully isolated saponin Quil A from the cortex of the South-American Tree Quillaja saponaria Molina [3,13–15] and found that Quil A stimulated both humoral and cellular immunity, as well as induced differential antibody isotypes [3, 6,15–17]. Since then, the Quil A has been commercialized and has gained widespread use in veterinary vaccines and pre-clinical studies [3,6,10,14,17–20]. Additional studies showed its effects when co-formulated with aluminum salts, liposomes and oil-in-water emulsions, and with amphipathic proteins and lipids forming detergent/lipid/saponin complexs termed immune-stimulating complexes (ISCOMs) [3,20–23]. However, Quil A is still a heterogeneous product, consisting of up to 23 different saponin peaks detectable by HPLC and its toxicity precluded its use in human vaccines [2–3,6,15,24]. Due to the fact that Quil A is a mixture, a further study was performed by Kensil et al. in 1991[2,6], in which 10 of RP-HPLC fractions from aqueous extract of Q. saponaria bark treated by ultrafiltration were tested and found the fractions QS-7, QS-17, QS-18 and QS-21 to be particularly potent. However, their toxicity varies considerably. QS-18, the major component of Q saponaria, was found to be highly toxic in mice, while QS-7 and QS- 21 shows far less toxicity [2,6,25]. QS-21 was further extensive studied because it is more abundant than QS-7 [7]. Studies showed that QS-21 promoted high antigen-specific antibody responses and CD8+ T-cell response in mice [2–6,17,26] and favored a balanced production of both IgG1 and IgG2a [1,2,27]; in contrast, aluminum hydroxide mainly promotes IgG1 production [2,6,27]. Further studies showed that QS-21 stimulated the production of cytotoxic T-lymphocytes (CTLs), induces Th1cytokines, interleukin-2 and interferon-gamma (IL-2 and IFN-γ), and antibodies of the IgG2a isotype to protein antigens [1,6,26,28,29]. A number of comparative studies has demonstrated that QS-21 significantly outperformed the other classes of adjuvants including glucan formulations, peptidoglycans, amphophilic block copolymers, bacterial nucleosides and bacterial lipopolysaccharides, in augmenting antibody responses as well as T-cell responses against target antigens [2,7]. Due to the high potency, QS-21 has been used as an effective adjuvant with a recombinant retroviral subunit vaccine against feline leukemia virus (FeLV), which is commercially available now [3,6,30]. However, the toxicity and the undesirable haemolytic effect still remain for human use [2,6,7]. QS-21 caused 50% haemolysis of sheep red blood cells (SRBC) at concentrations as low as 7–9 µg/ml (2, 29), and apparently, the side effects associated with QS-21 limit doses to about 50 µg with exception of cancer patient (melanoma, breast and prostate) [1,7]. It is clear that if the higher doses of this adjuvant could be safely used, its immunogenicity should be further increased [2,7]. The Adjuvant System (AS) developed by GlaxoSmithKline demonstrated that the apparent adverse events of QS-21 in rat skeletal muscle could be significantly reduced if QS-21 was formulated in AS01 (composed of liposomes, MPL and QS-21) at a dose of 5 µg, when compared to formulations containing QS-21 alone at the same dose, suggesting that the toxicity of QS-21 can be reduced when formulated with other appropriate adjuvants [5]. QS-21 containing adjuvants such as AS01, AS02 are currently tested in human clinical trials for various vaccine candidates and infectious agents, including influenza, malaria, hepatitis B virus (HBV), human papillomavirus (HPV), HIV/AIDS, tuberculosis (TB), non-small-cell lung carcinoma (NSCLC) and melanoma [1,4,5,8,17,28]. Over the past two decades, the saponin adjuvants have emerged as one of the leading adjuvant candidates. QS-21 containing complex adjuvants in particular have been widely popular in its application to develop vaccines platforms [4,5,7]. It is promising that these QS-21 containing complex adjuvants may be one of the first to be approved as alternative adjuvants in human use in the US in the future.

Immunomodulatory activity of isoflavones isolated from Iris kashmiriana: Effect on T-lymphocyte proliferation and cytokine production in Balb/c mice

This study deals with the evaluation of immunomodulatory action of a new isoflavone isonigricin (1) and first report isoirisolidone (2) isolated from rhizomes of Iris kashmiriana. Flowcytometric method was employed to study their effect on production of T-lymphocytes (CD4+ and CD8+ T-cells) and T-cell cytokines (IL-2, IL-4 and IFN-γ) in a dose-dependant manner. Drugs at doses of 0.025 to 0.8mg/kg per oral dose showed compound 1 to possess immunosuppressant activity on T-cells as well as cytokines, while compound 2 acted as immunostimulator for both cells and cytokines. However, their methylated products viz. 1OMe and 2OMe exhibited stimulatory effect on interleukins under study and suppressive effect on production of T-lymphocytes and IFN-γ. The oral LD50 in mice was observed to be more than 200mg/kg body weight. Such a difference in their activity is the manifestation of the position and nature of substituents present on isoflavonoidal skeleton and demonstrates the potential of these isolates as prospective immunomodulatory agents.

Remarkable stability of clinical grade reagents for gene-engineered T-lymphocyte production

Gene-engineered T-cell immunotherapy has emerged as a promising and potentially curative treatment for patients with numerous cancers, including renal cell carcinoma, metastatic melanoma, synovial cell sarcoma, colon cancer, neuroblastoma and multiple types of leukemia (1), and holds promise for many additional cancers and lifethreatening diseases, including HIV/AIDS. A major limitation to the widespread implementation of gene-engineered T-cell immunotherapy is the cost of obtaining current Good Manufacturing Practices (cGMP) clinical-grade reagents with which to prepare transduced cells. In this issue of Cytotherapy, Lamers et al. show the potential for long-term cold storage and continued use of multiple clinical-grade immunotherapy reagents, in some cases up to 14 years after their indicated expiration dates (2). In their article, the authors methodically evaluate the sterility and function of the T cell-stimulating anti-CD3 monoclonal antibody Orthoclone OKT3 (Janssen Biotech, Inc., Horsham, PA, USA), the widely used transduction reagent RetroNectin (TAKARA BIO INC., Shiga, Japan) and previously produced and qualified clinical-grade retroviral vector supernatant encoding a chimeric antigen receptor for the carbonic anhydrase IX protein (3). These results suggest that the shelf-lives of many required gene therapy transduction reagents can be significantly extended, provided that they are prepared as single-use aliquots according to cGMP standards and stored at 80 C. Although the needs for patient safety and qualityassured reagents are paramount, the prescribed dates of usage for reagents often fall within a short window,

Membrane Microdomains and Cytoskeleton Organization Shape and Regulate the IL-7 Receptor Signalosome in Human CD4 T-cells

Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center.

Effects of combined antiretroviral therapy on B- and T-cell release from production sites in long-term treated HIV-1+ patients

BackgroundThe immune system reconstitution in HIV-1- infected patients undergoing combined antiretroviral therapy is routinely evaluated by T-cell phenotyping, even though the infection also impairs the B-cell mediated immunity. To find new laboratory markers of therapy effectiveness, both B- and T- immune recovery were evaluated by means of a follow-up study of long-term treated HIV-1- infected patients, with a special focus on the measure of new B- and T-lymphocyte production.MethodsA longitudinal analysis was performed in samples obtained from HIV-1-infected patients before therapy beginning and after 6, 12, and 72 months with a duplex real-time PCR allowing the detection of K-deleting recombination excision circles (KRECs) and T-cell receptor excision circles (TRECs), as measures of bone-marrow and thymic output, respectively. A cross sectional analysis was performed to detect B- and T-cell subsets by flow cytometry in samples obtained at the end of the follow-up, which were compared to those of untreated HIV-1-infected patients and uninfected controls.ResultsThe kinetics and the timings of B- and T-cell release from the bone marrow and thymus during antiretroviral therapy were substantially different, with a decreased B-cell release and an increased thymic output after the prolonged therapy. The multivariable regression analysis showed that a longer pre-therapy infection duration predicts a minor TREC increase and a major KREC reduction.ConclusionsThe quantification of KRECs and TRECs represents an improved method to monitor the effects of therapies capable of influencing the immune cell pool composition in HIV-1-infected patients.

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn’s neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

Immunomodulatory activity of isoflavones isolated from Iris germanica (Iridaceae) on T‐lymphocytes and cytokines

The immunomodulatory activities of two isoflavones, 5,7-dihydroxy-6,4'-dimethoxyisoflavone (irisolidone) (1) and 5,4'-dihydroxy-6,7-methylenedioxyisoflavone (irilone) (2) isolated from Iris germanica (Iridaceae) is reported. Their influence on production of T-lymphocytes (CD4+ and CD8+ cells) and T-cell cytokines, namely Th1: IL-2, IFN-gamma and Th2: IL-4 and IL-5 in a dose-dependent manner was studied by flow cytometric method in Balb/c mice. Oral administration of drugs at doses of 0.1-0.8 mg/kg per oral dose showed 1 to possess stimulatory activity on T-cells and Th1 cytokine production, while as 2 acted as an immunosuppressant for both cells and cytokines. The methylated products of 1 and 2 showed a similar trend to that of their parent compounds but their activity was drastically decreased revealing the importance of free phenolic groups for their immunomodulating activities.