PHA Stimulation Research Articles

Fish Uses CTLA-4 Immune Checkpoint to Suppress mTORC1-Controlled T-Cell Glycolysis and Immunity.

As an immune checkpoint, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) suppresses the activation, proliferation, and effector function of T cells, thus preventing an overexuberant response and maintaining immune homeostasis. However, whether and how this immune checkpoint functions in early vertebrates remains unknown. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell response by CTLA-4 in bony fish. Tilapia CTLA-4 is constitutively expressed in lymphoid tissues, and its mRNA and protein expression in lymphocytes are upregulated following PHA stimulation or Edwardsiella piscicida infection. Blockade of CTLA-4 signaling enhanced T cell activation and proliferation but inhibited activation-induced T cell apoptosis, indicating that CTLA-4 negatively regulated T cell activation. In addition, blocking CTLA-4 signaling invivo increased the differentiation potential and cytotoxicity of T cells, resulting in an enhanced T cell response during E. piscicida infection. Tilapia CTLA-4 competitively bound the B7.2/CD86 molecule with CD28, thus antagonizing the CD28-mediated costimulatory signal of T cell activation. Furthermore, inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, c-Myc, or glycolysis markedly impaired the CTLA-4 blockade-enhanced T cell response, suggesting that CTLA-4 suppressed the T cell response of tilapia by inhibiting mTORC1/c-Myc axis-controlled glycolysis. Overall, the findings indicate a detailed mechanism by which CTLA-4 suppresses T cell immunity in tilapia; therefore, we propose that early vertebrates have evolved sophisticated mechanisms coupling immune checkpoints and metabolic reprogramming to avoid an overexuberant T cell response.

Expression of lymphocyte activation gene-3 on CD4+T cells is regulated by cytokine interleukin-18 in myasthenia gravis

Myasthenia gravis (MG) is a T cell-dependent, B cell-mediated, and complement-dependent autoimmune disease. Lymphocyte activation gene-3 (LAG-3; CD223) is an immune checkpoint protein that plays an important role in maintaining autoimmune tolerance and homeostasis. To investigate the cytokine-regulated expression pattern of LAG-3, CD4+T cells were sorted from the peripheral blood of healthy volunteers by density gradient centrifugation and stimulated with various cytokines in vitro. The expression of membrane LAG-3 (mLAG-3), membrane a disintegrin and metallopeptidase domain10 (mADAM10) and membrane ADAM17 (mADAM17) on CD4+T cells was detected by flow cytometry; the concentration of soluble LAG-3 (sLAG-3) was detected by ELISA; and the relative expression of genes at the transcriptional level was detected by fluorescence quantitative RT-PCR (qRT-PCR). sLAG-3 levels were significantly increased in the peripheral plasma of AChR Ab-positive patients with MG compared to healthy volunteers, while the percentage of mLAG-3 expression on CD4+T lymphocytes in the peripheral blood of patients with MG was significantly reduced. IL-18 inhibited mLAG-3 levels on CD4+T cells in a concentration-dependent manner. Additionally, the concentration of sLAG-3 in the supernatant increased. After PHA and IL-18 stimulation, ADAM10 and ADAM17 also increased compared to those in the PHA-active group. Moreover, there were significant differences in the expression of mADAM10 and mADAM17 in CD4+T lymphocytes between patients with MG and healthy volunteers. These results suggest that IL-18 may regulate the expression pattern of mLAG-3 in CD4+T cells and sLAG-3 via ADAM10- and ADAM17-mediated pathways, thus affecting the immune effects of CD4+T cells. This study provides a preliminary exploration of the upstream regulatory molecules of the LAG-3 and IL-18/LAG-3 signalling pathways for potential targeted therapy of autoimmune diseases in the future.

Expression and Immune Characterization of Major Histocompatibility Complex in Paralichthys olivaceus after Antigen Stimulation.

The Major histocompatibility complex (Mhc) is an important molecule for antigen presenting and binds to T cell receptors, activating T lymphocytes and triggering specific immune responses. To investigate the role of MhcII in adaptive immunity, in this study, mhcIIα and mhcIIβ of flounder (Paralichthys olivaceus) were cloned, polyclonal antibodies (Abs) against their extracellular regions were produced, respectively, and their distribution on cells and tissues and expression patterns, which varied by antigen stimulation or pathogen infection, were investigated. The results showed that the open reading frame (ORF) of mhcIIα is 708 bp, including 235 amino acids (aa); and the ORF of mhcIIβ is 741 bp, encoding 246aa. The mhcIIα and mhcIIβ were significantly expressed in gills, spleen, and peripheral blood leukocytes (PBLs). Their antibodies could specifically recognize eukaryotic expressed MhcIIα and MhcIIβ. MhcIIα+ and MhcIIβ+ cells were 30.2 ± 2.9% of the percentage in peripheral blood leukocytes. MhcII molecules were co-localized with CD83 and IgM on leukocytes, respectively, but not on CD4+ or CD8+ T lymphocyte subpopulations. The expression of both mhcIIα and mhcIIβ were significantly upregulated in flounder after bacteria and virus challenges. The percentages of MhcII+ cells, MhcII+/CD83+, and MhcII+/IgM+ double-positive cells increased significantly after PHA and ConA stimulation, respectively; they varied significantly in PBLs after polyI:C stimulation, and no variations were found after LPS treatment. In the meantime, variations in MhcII+ cells were consistent with that of CD4+ T lymphocytes. These results suggest that MhcII, mainly expressed in B cells and dendritic cells, play an essential role in antigen presentation, and respond significantly to exogenous antigens and T cell-dependent antigens. These results may provide an important reference for the study of cellular immunity in teleosts.

Larval T Cells Are Functionally Distinct from Adult T Cells in Xenopus laevis.

The amphibian Xenopus laevis tadpole provides a unique comparative experimental organism for investigating the roles of innate-like T (iT) cells in tolerogenic immunity during early development. Unlike mammals and adult frogs, where conventional T cells are dominant, tadpoles rely mostly on several prominent distinct subsets of iT cells interacting with cognate nonpolymorphic MHC class I-like molecules. In the present study, to investigate whole T cell responsiveness ontogenesis in X. laevis, we determined in tadpoles and adult frogs the capacity of splenic T cells to proliferate invivo upon infection with two different pathogens, ranavirus FV3 and Mycobacterium marinum, as well as invitro upon PHA stimulation using the thymidine analogous 5-ethynyl-2'-deoxyuridine and flow cytometry. We also analyzed by RT-quantitative PCR T cell responsiveness upon PHA stimulation. In vivo tadpole splenic T cells showed limited capacity to proliferate, whereas the invitro proliferation rate was higher than adult T cells. Gene markers for T cell activation and immediate-early genes induced upon TCR activation were upregulated with similar kinetics in tadpole and adult splenocytes. However, the tadpole T cell signature included a lower amplitude in the TCR signaling, which is a hallmark of mammalian memory-like T cells and iT or "preset" T cells. This study suggests that reminiscent of mammalian neonatal T cells, tadpole T cells are functionally different from their adult counterpart.

Quantification of camelid cytokine mRNA expression in PBMCs by microfluidic qPCR technology

Camelids are economically and socially important in several parts of the world and might carry pathogens with epizootic or zoonotic potential. However, biological research in these species is limited due to lack of reagents. Here, we developed RT-qPCR assays to quantify a panel of camelid innate and adaptive immune response genes, which can be monitored in a single run. The assays were validated with PHA, PMA-ionomycin, and Poly I:C-stimulated PBMCs from alpaca, dromedary camel and llama, including normalization by multiple reference genes. Further, comparative gene expression analyses for the different camelid species were performed by a unique microfluidic qPCR assay. Compared to unstimulated controls, PHA and PMA-ionomycin stimulation elicited robust Th1 and Th2 responses in PBMCs from camelid species. Additional activation of type I and type III IFN signalling pathways was described exclusively in PHA-stimulated dromedary lymphocytes, in contrast to those from alpaca and llama. We also found that PolyI:C stimulation induced robust antiviral response genes in alpaca PBMCs. The proposed methodology should be useful for the measurement of immune responses to infection or vaccination in camelid species.

Влияние хронического облучения на концентрацию белка FOXP3 в лизатах митоген-стимулированных мононуклеаров крови

Disruptions of the Treg differentiation and functioning processes can play one of the crucial roles in the pathogenesis of radiation-induced malignant neoplasms in residents of the Techa riverside villages, who were chronically exposed in the low-to-medium dose range with predominant damage to the red bone marrow (RBM). This study aimed to determine the effect of radiation exposure, gender, age at the time of examination, and ethnicity on concentration of FOXP3 protein in lysates of mitogen-stimulated peripheral blood mononuclear cells in chronically exposed individuals in the period of cancer effects development. The main group consisted of 30 people aged 67–80 years, predominantly female and Turks. The comparison group included 10 unexposed individuals of similar age, gender, and ethnicity. In the main group, the mean dose to RBM was 867 mGr, to the thymus and peripheral lymphoid organs — 125 mGr. After 24-hour in vitro PHA stimulation, mononuclears were lysed, and the concentrations of the total protein and FOXP3 (using quantitative enzyme immunoassay) were measured. Among the different dose groups, there were no significant differences in FOXP3 concentration in mitogen-stimulated mononuclears (prior to the stimulation: 0 pg/ml in the comparison group and 3.50 ± 1.50 (0–27.19) pg/ml in the main group at p = 0.349; after the stimulation, respectively: 1.54 ± 1.51 (0–15.16) pg/ml and 9.71 ± 3.86 (0–77.92) pg/ml, p = 0.512). The variability of individual values is slightly higher in the main group than in the comparison group. Preliminary results allow concluding that the dose to RBM, thymus and peripheral lymphoid organs, age at the time of examination, gender, and ethnicity have no statistically significant effect on the concentration of FOXP3 protein in the lysates of the mitogen-stimulated peripheral blood mononuclear cells of chronically exposed people.

Assessment of in Vitro Immunomodulatory Activity of Ethanolic Extract of Crataeva Nurvula Buch. Ham. Bark

The current study was carried out to assess the immunomodulatory activity of Ethanolic extract Crataeva nurvula bark (EECNB) using in vitro methods. In vitro immune modulatory activity was investigated by cellular lysosomal enzyme assay and phagocytosis evaluation. The release of cellular lysosomal enzyme was found to be significant (p<0.05) at 200 μg/ml (Stimulation Index = 1.982) and 100 μg/ml (S.I= 1.564) of EECN with respect to control PHA (Stimulation Index =1.00) maximum phagocytic activity of EECNB was evident at 500g/ml and With gradual increase in the dilutions of the plant extract there was a corresponding decrease in phagocytic activity. The present has demonstrated the immunomodulatory potential of EECNB (as it has good stimulation index and maximum phagocytic activity) based on the results of lysosomal and phagocytosis assay. Further studies are needed to support its immonumodulatory activity.

The Effects of A Short Period of A Ketogenic Diet on Blood Biochemistry and Immunological Status in Overweight Thai Subjects

Being overweight and obese is a fundamental health problem worldwide and is associated with many diseases. The ketogenic diet (KD) includes high fat and moderate protein, with the daily consumption of carbohydrates amounting to less than 10 % daily intake. KD is a popular choice for weight loss but has benefited many diseases, such as metabolic diseases and neurodegenerative disorders. Nonetheless, the effect of KD on overweight but healthy individuals is limited. Therefore, the objectives of the present study were to evaluate the effects of the KD program on the physical, blood biochemistry, hematological and immunological status of overweight, healthy individuals. The 9 overweight Thai women with body mass index (BMI) values over 25 kg/m2 participated in a self-conduct KD program for 28 days. The mean age of the group was 28.44 years. With 28-days of KD, a significant (p < 0.01) increase in blood ketone confirmed the ketosis status of participants. The mean blood ketone concentration increased from 0.41 ± 0.061 to 1.22 ± 0.267 mmol/L. Most of the participants’ physical parameters, such as body mass index (BMI), body weight, chest circumferences, hip circumferences, and arm circumferences, significantly decreased after 28-days of KD. Furthermore, the biochemistry indices (total cholesterol, triglyceride, LDL-C, and HDL-C) did not significantly differ from the baseline. However, fasting blood sugar decreased (p < 0.01) from 88.00 ± 1.014 to 83.78 ± 1.289 mg/dL. KD did not influence all hematological and immunological indices such as nitric oxide (NO) production (unstimulated and in 2.5 µg/mL PHA stimulation) and T cell subpopulations. Nonetheless, it increased NO production in 5 µg/mL PHA stimulation and mononuclear cell proliferation capacity (proliferation index). The data revealed that short-term KD in this study decreased participants’ physical body compositions and blood glucose levels. Moreover, the immune status after KD proved the immunomodulation effect of KD. HIGHLIGHTS Thai overweight subjects whose self-conducted 28-days ketogenic diet demonstrated Significantly decreased the bodyweight Significantly decreased the body compositions Significantly decreased blood sugar level. Significantly affected immune systems by increasing immune cell proliferation capacity and responding to the non-specific stimulator GRAPHICAL ABSTRACT

Molecular Characterization and Expression Analysis of Novel Interleukin-1 Family Member (nIL-1Fm) Gene in Nile Tilapia (Oreochromis niloticus)

In this study, a novel interleukin-1-family member (nIL-1Fm) was studied from the Nile tilapia (Oreochromis niloticus), a widely cultured fish species in brackish water and freshwater in Thailand and worldwide. The complete sequence of the Nile tilapia nIL-1Fm comprised 2474 bp, encoding 350 amino acids. The genome organization of nIL-1Fm had seven exons and six introns, which were conserved in teleost fish. During tissue distribution analysis, the nIL-1Fm was extremely increased in the head kidney, tailfin and trunk kidney, which had the highest levels, followed by the spleen. Additionally, nIL-1Fm was significantly up-regulated under PAMP stimulation, whereas the stimulation of LPS, PHA and poly I:C could significantly induce nIL-1Fm expression in vitro. Moreover, nIL-1Fm transcription was significantly induced when infected with Streptococcus agalactiae in the spleen, liver and intestine. Taking together, our results indicated that nIL-1Fm can expand our knowledge of the IL-1 cytokine family in fish immune response.

PB2295: EVANS SYNDROME AND PHENOTYPIC HETEROGENEITY OF SASH3 GERMLINE LOSS-OF-FUNCTION MUTATIONS BEYOND X-LINKED IMMUNODEFICIENCY

Background: “Evans syndrome (ES)” denotes a heterogeneous disease entity, defined by two or more autoimmune cytopenias, due to mostly unknown pathogenic mechanisms. Further, ES is increasingly recognized in pediatric patients with defined inborn errors of immunity (IEIs) including pathogenic variants in CTLA4, STAT3, TNFRSF6, PIK3CD, and KRAS. Loss-of-function mutations in Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3) have recently been reported as a novel IEI entity in four patients with X-linked combined immunodeficiency with immune dysregulation including immune cytopenias. SASH3 is an X-chromosomal encoded adaptor protein that is exclusively expressed in lymphocytes. Although knowledge of the exact molecular function of this protein is scarce, humans and mice lacking expression of SASH3 show defects in proximal and distal T-cell receptor signalling. Aims: We aimed to dissect aberrant immune cell functionality in SASH3-deficient patients to further enhance our understanding of pathogenic mechanisms in patients with ES. Methods: We performed whole exome sequencing, deep clinical phenotyping and immunophenotyping in a patient with ES and his clinically asymptomatic brother. Results: Our index patient presented with severe ES and recurrent infections at the age of 8 years. Immune phenotyping of his peripheral mononuclear cells (PBMCs) showed transient CD4+ T- and B-cell lymphopenia and reduced T-cell proliferation upon PHA and SEB stimulation. Furthermore, the patient had hypogammaglobulinemia, and the ‘transitory CD21 low B-cells’ were significantly increased, reflecting impaired lymphocyte maturation possibly causing the autoreactive phenotype of the disease. Interestingly, the patient developed large hemorrhagic splenic cysts during ITP associated bleeding diathesis, and thus underwent splenectomy, which led to a complete remission of ES. Using whole-exome sequencing, we discovered a germline nonsense mutation in SASH3 (c.862C>T;p.Arg288Ter) in a now 16 years-old patient with ES. Surprisingly, this maternally inherited mutation was also identified in the hitherto clinically inconspicuous younger brother. The identified mutation is identical to the one reported in two males in the discovery of SASH3 deficiency. Divergently, the now 8-year-old brother who is also carrying the same variant has no clinically apparent phenotype until now. Notably, disease onset occurred earlier (i.e., between age 2 and 5 years) in the previously reported patients compared to our patients. Summary/Conclusion: We present the 5th and 6th patient with germline hemizygous deleterious variants in SASH3. Thus, this study expands the clinical phenotype and underlines a marked phenotypic heterogeneity as illustrated in siblings bearing the same mutation. Future studies will need to address comprehensively the phenotypic spectrum of the disease and genetic and non-genetic modifiers of clinical phenotypes and severity.

The Expression of CD28 and Its Synergism on the Immune Response of Flounder (Paralichthys olivaceus) to Thymus-Dependent Antigen.

CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder (Paralichthys olivaceus); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated in vitro. fCD28 consists of four exons and three introns, and the full-length cDNA of fCD28 was 675-bp encoded 224 amino acids. The conserved motif (121TFPPPF126) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of fCD28 is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured in vitro with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of interleukin-2 (IL-2) and nuclear factor of activated T cells (NFAT) in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation.

Genetic variation in NDFIP1 modifies the metabolic patterns in immune cells of multiple sclerosis patients

One of the 233 polymorphisms associated with multiple sclerosis (MS) susceptibility lies within the NDFIP1 gene, and it was previously identified as eQTL in healthy controls. NDFIP1 shows interesting immune functions and is involved in the development of the central nervous system. We aimed at studying the NDFIP1 variant on activation and metabolism of immune cells. NDFIP1 mRNA and protein expression were assessed in PBMCs by qPCR and western blot in 87 MS patients and 84 healthy controls genotyped for rs4912622. Immune activation after PHA stimulation was evaluated by CD69 upregulation, and metabolic function of both basal and PHA-activated lymphocytes was studied by Seahorse Xfp-Analyzer. In minor-allele homozygous controls but not in patients, we found higher NDFIP1 expression, significantly reduced protein levels, and CD69 upregulation in B- and T-cells. PBMCs from minor-allele homozygous controls showed significantly higher basal mitochondrial respiration and ATP production compared to major-allele carriers, while minor-allele homozygous patients showed significantly lower metabolic activity than carriers of the major allele. In conclusion, we describe associations in minor-allele homozygous controls with lower levels of NDFIP1 protein, CD69 upregulation, and raised mitochondrial activity, which are not replicated in MS patients, suggesting a NDFIP1 differential effect in health and disease.

Dairy Fat Consumptions Do Not Modulate Immune Functions in a Swine Model of Insulin Resistance: A Preliminary Analysis

ObjectivesBiomarkers of full-fat dairy consumption have been inversely associated with variables of insulin resistance (IR) and immune function. To understand the effect of consuming dairy fat per se on immune function in the context of IR, we used our established low birthweight (LBW) swine model of high fat diet induced IR to compare regular fat dairy products vs. low-fat dairy products and compared to a control high fat diet (CHF). We hypothesize that consuming a diet rich in dairy fat will improve immune function in the context of IR. MethodsAt 5 weeks of age, LBW piglets were randomized to consume one of the 3 experimental diets: 1) CHF, 2) HF supplemented with 3 servings high fat dairy (HFDairy) and 3) HF supplemented with 3 servings low fat dairy (LFDairy). As comparison groups, normal birthweight (NBW) piglets were fed a CHF or standard pig grower diet (Chow). A total of 35 pigs (LBW-CHF n = 8, LBW-HFDairy n = 8, LBW-LFDairy n = 8, NBW-CHF n = 6, NBW-Chow n = 5) were fed for 7 weeks. At 12 weeks of age, pigs were euthanized for tissue and blood collections. Mitogen stimulations on peripheral blood mononuclear cells were conducted to assess immune responses. ResultsResults show that there were no statistical differences in IL-2, IL-10 and TNF-α levels after pokeweed mitogen (PWM, T and antigen presenting cell mitogen) and phytohaemagglutinin (PHA, T cell mitogen) stimulations between all LBW groups. However, IL-10 levels after PHA stimulation were found to be higher in NBW-Chow compared to LBW-CHF, LBW-HFDairy and LBW-LFDairy (all P < 0.05). Similarly, IL-2 levels after PWM stimulation were found to be higher in NBW-Chow compared to LBW-CHF and LBW-HFDairy (both P < 0.05) groups but not LBW-LFDairy. ConclusionsCurrent data suggest that consumption of dairy products, regardless of the fat content, as little effect on immune function in the context of IR. However, we demonstrated that diet-induced IR piglets exhibit altered immune responses to a T cells mitogen, compared to NBW piglets. Funding SourcesAgriculture Funding Consortium, NSERC Discovery Program, Dairy Farmers of Canada-Nutrition Research Program.

An atypical KLRG1 in Nile tilapia involves in adaptive immunity as a potential marker for activated T lymphocytes

Killer cell lectin-like receptor G subfamily 1 (KLRG1) is a receptor generally expressed on effector CD8+ T cells or NK cells at terminal differentiation stage, and it will be highly induced for lymphocyte cytotoxicity upon pathogen infection or lymphocyte activation. However, little is known about the character or function of KLRG1 in lower vertebrates. In present study, we reappraised a molecule that previously defined as KLRG1 in the genomic sequence of Nile tilapia Oreochromis niloticus, and identified it as an atypical KLRG1-like molecule (defined as On-KLRG1-L), and illustrated its potential function serving as a marker representing effector T lymphocytes of fish species. On-KLRG1-L consists of two C-type lectin-like domains (CTLDs) without transmembrane region, and the tertiary structure of the CTLD is highly alike to that in mouse KLRG1. As a CTLD-containing protein, the recombinant On-KLRG1-L could bind PGN and several microbes in vitro. On-KLRG1-L was widely expressed in immune-associated tissues, with the highest expression level in the gill. Once Nile tilapia is infected by Aeromonas hydrophila, mRNA level of On-KLRG1-L in spleen lymphocytes were significantly up-regulated on 5 days after infection. Meanwhile, On-KLRG1-L protein was also induced on 5 or 8 days after A. hydrophila infection. Furthermore, we found both mRNA and protein levels of On-KLRG1-L were dramatically enhanced within several hours after spleen lymphocytes were activated by T cell-specific mitogen PHA in vitro. More importantly, the ratio of On-KLRG1-L+ T cells was also augmented after PHA stimulation. The observations suggested that the KLRG1-like molecule from Nile tilapia participated in lymphocyte activation and anti-bacterial adaptive immune response, and could serve as an activation marker of T lymphocytes. Our study thus provided new evidences to understand lymphocyte-mediated adaptive immunity of teleost.

A streamlined proliferation assay using mixed lymphocytes for evaluation of human mesenchymal stem cell immunomodulation activity

BackgroundMesenchymal stromal cells (MSCs) have been proposed for treatment of acute respiratory distress syndrome (ARDS), graft versus host disease (GVHD), wound healing and trauma. A consensus is building that immunomodulation by MSCs is important for therapeutic potential. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, potentially reflecting an ability to suppress PBMC inflammatory responses in vivo. Current mixed lymphocyte reaction (MLR) assays commonly used to evaluate MSC potency generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated PBMCs in the presence of mitotically inactive MSCs. Proliferation of PBMCs is monitored by several methods, including incorporation of radiolabeled nucleotides, BRDU labeling and ELISA assay or flow cytometry of carboxyfluorescein labeled PBMCs. Here we present a streamlined assay using MSCs in a direct co-culture system with unmodified MSCs using a luminescent ATP assay to evaluate both PBMC and MSC proliferation/survival. MethodsPBMCs were isolated from fresh anti-coagulated whole blood by centrifugation over Ficoll-Paque in LeucoSep tubes. Isolated PBMCs from 8 to 10 donors were pooled and cryopreserved at 1 × 107/ml in 50% RPMI medium,10% DMSO, 40% human AB serum. MSCs derived from bone marrow, adipose tissue or umbilical cord (BM-MSC, Ad-MSC, UC-MSC, respectively) were serially diluted starting at 50–60,000 cells/well and cultured in 96 well plates for 4–48 h in their respective medium. On Day 0, MSCs were washed, resuspended in PBMC media (RPMI with 10% FBS, 2 mM Glutamine, 10 mM HEPES, pH 7.4) and incubated with or without 150,000 freshly thawed pooled PBMCs/well, in the presence or absence of phytohemagglutinin A (PHA, 0–5 μg/ml). Proliferation of both MSCs (adherent) and PBMCs (non-adherent) was assessed by quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo (Promega). Culture supernatant contained PBMC, while washed adherent cells were primarily MSCs. Both cell types were incubated for 30 min with an equal volume of Cell Titer-Glo reagent and then assayed in white plates on a luminescence plate reader. ResultsPBMC proliferation in response to PHA stimulation resulted in a robust increase in ATP by 72 h, with >6 fold increase over unstimulated PBMCs, which showed no increase. MSC proliferation was decreased <20% at the highest PHA concentrations. Co-culture with MSCs suppressed PBMC proliferation dependent upon MSC passage number, source, and prior growth conditions. Total time to complete the ATP assay was under an hour including incubations. With minimal manipulations in the assay, intra- and inter- assay variations averaged 11.1 and 15.7% respectively. ConclusionsDirect co-culture of live unmodified MSCs with freshly thawed pooled PBMCs gives a robust determination of immunosuppression by MSCs with unparalleled ease. Graded responses can be determined, allowing comparison of potency between MSC preparations as in comparisons between freshly thawed and cultured MSCs as well as interferon-γ licensed MSCs. With the 96 well plate assay, far fewer PBMCs are generally required than in a typical flow cytometry determination. This streamlined assay can be performed within 72 h, without irradiating cells and without specialized equipment.

Evaluation of Maternal Safety Following Prenatal Cell and Gene Therapy for Hemophilia a in a Large Animal Model Demonstrates Absence of Maternal Exposure to the Cells or Gene Products Infused into the Fetus

Prenatal transplantation (PNT) using stem/progenitor cells has been performed in more than 50 patients using an ultrasound-guided approach, proving that this procedure poses minimal risk to both the fetus and the mother. Since 75% of hemophilia A (HA) patients have a family history of HA, and prenatal diagnosis is available to pregnant HA carriers in most western countries, PNT constitutes a clinically viable approach to treat these patients. In addition, the type of mutation and family history can predict the severity of the disease and the patient's likelihood of developing FVIII inhibitors, thus allowing intervention in those who could most benefit from this approach. Nevertheless, maternal safety is a critical consideration for PNT, particularly with respect to possible exposure to the cells or gene products produced by the cells infused into the fetus. We have previously demonstrated that transplanting sheep fetuses (n=23) with human placental cells (PLC) transduced with a lentiviral vector encoding a bioengineered fVIII transgene (mcoET3), at a dose of 107-108 cells/kg, increased plasma fVIII activity levels for at least 3 years after transplant. Therefore, here we studied ewes (n=4) during pregnancy and those who had given birth (n=6) to lambs treated in utero. The latter were assessed longitudinally over a course of 5 months, ranging from 18 to 32 months after delivery of a PNT recipient. First, we investigated whether any of the ewes, during pregnancy or at any point thereafter,developed an immune response to the transplanted product, PLC-mcoET3. Peripheral blood mononuclear cells (PBMC) harvested from the different ewes readily proliferated in response to human PBMC and upon PHA stimulation, but PLC-mcoET3 and non-transduced PLCs did not elicit an immune response from PBMC from any of the ewes, demonstrating that PNT did not immunize the ewe to the transplanted cells. We also used an mcoET3-specific ELISA to investigate whether the ewes developed antibodies to the mcoET3 protein. At no time point were ewes ever found to harbor anti-mcoET3 IgGs in their plasma. To determine whether mcoET3-specific T effector cells developed in the ewes, we performed ELISpot assays using PBMC collected during pregnancy and at various time points thereafter, and no mcoET3-specific Th1 or Th2 cells were detected in these ewes. To assess the potential circulation and/or engraftment of PLC-mcoET3 in the ewes during pregnancy or at any time after delivery, maternal PBMC collected during pregnancy, and maternal-side placental tissue and umbilical cord tissue (UBC) collected at birth were analyzed using RT-qPCR (detection limit &amp;lt;0.01%) with primers specific for human FVIII (produced constitutively by the PLC) and mcoET3. Maternal PBMC, UBC, and placental tissue were all devoid of PLC-mcoET3, as evidenced by the complete absence of mcoET3 and human FVIII mRNA. Histopathologic analysis of the placental tissue demonstrated no abnormalities or ectopic tissue formation. Overall, these studies suggest that the administration of PLC-mcoET3 to the fetus during gestation does not result in detectable maternal exposure to the cells or gene products, attesting to the safety of this approach. Disclosures Doering: Kilpatrick, Townsend &amp; Stockton: Consultancy; Expression Therapeutics, LLC: Current equity holder in private company, Patents &amp; Royalties, Research Funding.

Murine transfer of a human gene variant associated with exceptional longevity displays senolytic effects both in immune compartment and endothelium of aged mice

Abstract The persistence and accumulation of senescent cells has been shown to potentially play a role in the pathophysiology of age-related cardiovascular diseases. Indeed with time, a decline in immune efficacy, termed immunesenescence, and a deleterious secretory phenotype of senescent cells (SASP) generate that inflammatory background mainly mediating the elderly cardiovascular phenotype. Long Living Individuals (LLI) which delay aging, represent a model of positive biology and an exceptional resource to study and find a way to improve general public health. Previous studies from our group have shown that a human gene associated with exceptional longevity (LAV-BPIFB4) was able to block the atherosclerotic process in ApoE−/− mice by conferring the animals with a pro-resolving M2 macrophages profile. Furthermore, LAV-BPIFB4 promotes the recruitment of hematopoietic stem cells, reparative vascularization and frailty reduction. Here we sought to underpinn the role of LAV-BPIFB4 in counteracting the age-related remodeling of the immune responses. The effects of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the immune dynamics in old mice have been investigated by an extensive flow cytometric approach in lymphoid tissues (bone marrow, spleen and peripheral blood). C57BL/6J mice were assigned to two age-matched experimental groups: a treatment group (AAV-LAV-BPIFB4; N=6 mice, aged 18–23 months and a control group (AAV-GFP; N=6 mice, aged 18–23 months. 30th and 60th day since the beginning of the infection, SA-beta Gal substrate has been used to identify CD45+ senescent cells in freshly isolated PBMC, splenocytes, bone marrow (BM)-derived cells. As expected, we monitored an increase in SA-betaGal activity in blood. This increase is most significant in CD11b+ myeloid cells, whithout affecting neither CD3+T neither NK1.1+Natural Killer (NK) cell compartment. Notably 30 days AAV-LAV-BPIFB4 infection and to a more the 60 days-treatment, resulted in a significant decrease in senescent pool of peripheral immune cells and a concomitant enrichment of senescent cells in spleen. Concomitantly, aorta from AAV-LAV treated mice showed significant reduction in SA-beta Gal positive area. Furthermore a LAV-BPIFB4 induction of pro-resolving M2 macrophages compared to control group was documented in the main haemocateretic organ. As consequence the senolytic effect of LAV-BPIFB4 gene-therapy well correlated with the rescue of proliferative index of splenocytes upon PHA stimulation, and their functional protective response to lipopolysaccharide (LPS) in term of IL-6 and TNF-alpha secretion. The restoration of a protective and balanced immune response finally reflected the reduction of senescent phenopype acquired by mouse aortic endothelial cells during the aging process in vivo. A better underpinning of the senolytic action of LAV-BPIFB4 may offer a valuable therapeutic tool to reverse aging phenotype causing most of cardiac diseases Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Cariplo Foundation (n.2016-0874) to AAP and CV

PHA stimulation may be useful for FDXR gene expression-based biodosimetry.

Objective(s):Nowadays, ionizing radiation (IR) has a significant contribution to the diagnostic and therapeutic medicine, and following that, health risks to individuals through unexpected exposure is greatly increased. Therefore, biological and molecular technology for estimation of dose (biodosimetry) is taken into consideration. In biodosimetry methods stimulation of cells to proliferation is routine to achieve more sensitivity of techniques. However, this concept has recently been challenged by new molecular methods such as gene expression analysis. This study aims to investigate the stimulation effects on gene expression biodosimetry.Materials and Methods:The blood samples were taken from15 patients who were irradiated by TC-99 MIBI, before radiopharmaceutical injection and 24 hr after injection. Lymphocytes were extracted immediately and activated by (phytohemagglutinin) PHA for 24 hr and XPA and FDXR expression levels were investigated by employing relative quantitative Real-Time PCR.Results:The results of this study show a significant increase in the FDXR expression level and a significant decrease in the XPA after stimulation of irradiated lymphocytes. Interestingly, a significant increasing trend in the FDXR expression level (at 0.05 significance level) following cell stimulation to the division was observed.Conclusion:Our results suggest that the PHA activation role in gene expression-based biodosimetry is strongly depended on the target genes and the relevant protein pathways. Finally, cell stimulation looks to be useful for some specific genes, such as FDXR, due to the increasing trend in expression and improvement of sensitivity of gene expression-based biodosimetry method.

T-Cell Reconstitution after Unrelated Donor HSCT Using Immunotherapy with CD25/71 Allodepleted Donor T Cells: Results of the Randomised Icat Study

Karl S Peggs and Sarah J Albon contributed equally to the work and are joint first author Introduction Alemtuzumab reduces the incidence of GVHD after unrelated donor stem cell transplant (MUD SCT) but delays immune reconstitution resulting in high morbidity/mortality from viral infections. Previous studies have suggested that adoptive transfer of allodepleted donor T cells (ADTs) improves immunity after SCT but this has never been tested in a randomised study. We developed a methodology for selective immunomagnetic depletion of alloreactive T-cells upregulating CD25 and CD71 after activation with host dendritic cells (DC) and showed that ADTs retain anti-viral responses with minimal host alloreactivity (Samarasinghe et al Blood 2010). We have now tested whether ADTs can safely be used to improve immune reconstitution after MUD SCT for haematological malignancies in a randomised Phase II multi-centre clinical study; ICAT (NCT01827579). Methods Patients undergoing Alemtuzumab-based peripheral blood SCT from a 9/10 or 10/10 MUD for haematological malignancy were randomised 2:1 to receive either the ATIMP (ADTs) or standard of care. Two weeks prior to SCT, patients randomised to ATIMP underwent a leucapheresis from which DCs were generated. Irradiated patient-derived DCs were then co-cultured with peripheral blood mononuclear cells (PBMC) from an unstimulated leucapheresis/500ml blood draw from the donor to activate alloreactive T cells. Four days later, the co-culture was depleted of CD25+ and CD71+ fractions by immunomagnetic depletion on the CliniMACs, sampled for residual alloreactivity and sterility, and cryopreserved. Patients randomised to the ATIMP were scheduled to receive 3 escalating doses of ADTs (0.1x106/Kg at day 30, 0.3x106/Kg at day 60 and 1x106/Kg at day 90 post-SCT) until either there was &gt;grade 1 aGVHD or they had normal circulating T cells (&gt;700/µL). The primary end-point of the study was circulating CD3+ T cell count at 4 months post-SCT with one-sided 15% significance level. Acute/chronic GVHD were graded using the Seattle/NIH criteria respectively. Results Twenty one patients were treated, 13 on the ATIMP arm and 8 on the control arm. The median age was 53 years and 67% (14) were male. 12 were AML/Myelodysplasia, 5 NHL, 3 CLL/CML and 1 HL. The median follow-up time is 14 months. Five of 13 ATIMP patients received 1 dose of ADTs, 4/13 2 doses and 4/13 all 3 doses. The incidence of acute and chronic GVHD was comparable between the arms. Overall, 7/13 ATIMP patients developed significant (&gt;Grade 2) acute GVHD compared to 4/8 of the control arm (p&gt;0.99). 3/13 patients in the ATIMP arm and 2/8 patients in the control arm developed severe aGVHD (all Grade 3). Three of 13 ATIMP cohort patients developed chronic GVHD (1 mild, 1 moderate, 1 severe), compared to 3/8 (all mild) in the control cohort. At 4 months, the circulating CD3+ T cell count mean was 730/µL (range 10-4080) in the ATIMP group and 212.5/µL (range 10-500) in the control group (1-sided p=0.11). However, the data was not normally distributed (Wilcoxon 1-sided p=0.18). Three ATIMP patients had high CD3+ T cell count at 4 months (&gt;1000/µL). At 6 months, the mean circulating CD3+ T cell count was 833.6/µL (range 20-2690) and 327.5/µL (range 10-860). At month 4, the mean PHA stimulation index in the ATIMP arm was 16.8 (range 0.67- 73.1) vs 3.8 (range 1.1-8.2) in the control group. At 4 and 6 months post-SCT, spectratyping analysis showed no evidence of a difference in Vβ diversity between the 2 arms in both CD4+ and CD8+ cells. The 1-year survival rate in the ATIMP cohort is 92% vs 88% in the control, and 1-year disease free survival rate 67% in the ATIMP cohort vs 70% in the control. Conclusions These data suggest that adoptive transfer of ADTs improves T cell reconstitution in some patients after MUD SCT and that the GVHD rates were similar between ATIMP and control groups. Figure 1: Kinetics of T cell recovery after transplant in ATIMP (blue) and Control (red) patients. Mean +/- SEM shown. Figure 1 Disclosures Peggs: Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees. Ghorashian:UCLB: Patents &amp; Royalties: UCLB; Celgene: Honoraria; novartis: Honoraria. Amrolia:UCLB: Patents &amp; Royalties.

Identification, molecular characterization and functional analysis of interleukin (IL)-2 and IL-2like (IL-2L) cytokines in sea bass (Dicentrarchus labrax L.)

In mammals, interleukin (IL)-2, initially known as a T-cell grow factor, is an immunomodulatory cytokine involved in the proliferation of T cells upon antigen activation. In bony fish, some IL-2 orthologs have been identified, but, recently, an additional IL-2like (IL-2L) gene has been found. In this paper, we report the presence of these two divergent IL-2 isoforms in sea bass (Dicentrarchus labrax L.). Genomic analyses revealed that they originated from a gene duplication event, as happened in most percomorphs. These two IL-2 paralogs show differences in the amino acid sequence and in the exon 4 size, and these features could be an indication that they bind preferentially to different specific IL-2 receptors. Sea bass IL-2 paralogs are highly expressed in gut and spleen, which are tissues and organs involved in fish T cell immune functions, and the two cytokines could be up-regulated by both PHA stimulation and vaccination with a bacterial vaccine, with IL-2L being more inducible. To investigate the functional activities of sea bass IL-2 and IL-2L we produced the corresponding recombinant molecules in E. coli and used them to in vitro stimulate HK and spleen leukocytes. IL-2L is able to up-regulate the expression of markers related to different T cell subsets (Th1, Th2 and Th17) and to Treg cells in HK, whereas it has little effect in spleen. IL-2 is not active on these markers in HK, but shows an effect on Th1 markers in spleen. Finally, the stimulation with recombinant IL-2 and IL-2L is also able to induce in vitro proliferation of HK- and spleen-derived leukocytes. In conclusion, we have demonstrated that sea bass possess two IL-2 paralogs that likely have an important role in regulating T cell development in this species and that show distinct bioactivities.