Number Of IFN-γ Producing Cells Research Articles

Adenoviral delivery of soluble ovine OX40L or CD70 costimulatory molecules improves adaptive immune responses to a model antigen in sheep.

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L- CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.

Reduced number of IFN-γ producing cells in peripheral blood is a biomarker for patients with renal cell carcinoma.

Renal cell cancer (RCC) is the most lethal of all the common urologic cancers and constitutes 2.2% of all malignancy diagnoses. The incidence of RCC has been steadily increasing in recent decades. The classic risk factors of RCC include smoking, hypertension, obesity, genetics, and genetic mutations. Recent studies also revealed that RCC was an immunogenic tumor and affected by host immune status. Among the pan‐cance, RCC presented with the highest degree of immune infiltration, indicating RCC patients might benefit from immunotherapy. A new immune classification of RCC has been developed by Su et al. based on tumor‐infiltrating lymphocytes to guide clinical practice. However, these studies mainly focus on biomarkers derived from tumor microenvironment (TME), the biomarkers based on peripheral blood samples to RCC have rarely been described. We collected peripheral blood samples from RCC patients and their matched healthy controls and detected the number of IL‐2 and IFN‐γ producing cells by implementing an enzyme‐linked immunospot (ELISPOT) assay. This is the first study to report blood‐based immune biomarkers for RCC using an ELISPOT assay. Our results suggested the frequency of IFN‐γ producing cells but not IL‐2 producing cells was associated with RCC risk. These findings warrant further validation in larger prospective studies.

Abstract P2-14-11: Immune response monitoring in breast cancer patients treated with neoadjuvant chemotherapy combined with dendritic cell vaccines

Abstract INTRODUCTION. Breast cancer (BC) is one of the most frequent cancers in women worldwide. Lack of therapeutic efficacy in some BC subtypes makes it necessary to develop new strategies. It is known that immune system plays a key role in the tumour control. Moreover, BC is immunogenic and the intensity of the immune response influences the clinical efficacy, and is correlated with a good prognosis. For that, manipulating the immune system to make it more effective could be an option. We design a clinical trial (NCT01431196) to evaluate the clinical efficacy of the combination of standard treatment with monocyte-derived dendritic cells vaccines preloaded with autologous tumour lysate (DCV) in stage I-III BC patients without HER2 overexpression. An in-depth immunological study was carried out. OBJECTIVES. To evaluate the immune response induced by the combination of neoadjuvant chemotherapy ± DCV ± radiation ± endocrine therapy in BC patients. METHODS. Serum and peripheral blood mononuclear cells (PBMCs) were collected from 20 luminal and triple negative BC patients before and after treatment. In serum, the presence of anti-tumour antibodies was studied by flow cytometry. PBMCs phenotype was evaluated by flow cytometry. In addition, functional studies were performed by incubating PBMCs with dendritic cells preloaded with autologous tumour lysate to evaluate specific immune response; specifically, T cell proliferation assay by [3H] thymidine incorporation and IFN-γ-producing cell assays by ELISPOT were done. Finally, the TCR clonality was assessed by flow cytometry. RESULTS. We identified IgG and IgM antibodies specific for breast cancer cell lines in the serum of 20 % and 40% of patients. Myeloid-derived suppressor cells decreased and NK cells increased with the treatment. Moreover, activation markers, such as HLADR, significantly increased in CD4+ and CD8+ lymphocytes after treatment, whereas PD1 and TIM3 decreased. In addition, an increase in the proliferation of specific T cells and in the number of IFN-γ producing cells after stimulation with tumor lysate pulsed DC was detected after treatment. Finally, very good clinical responders (pT4/5 with Miller&Payne classification and pN0) had higher TCR diversity index (DI) in CD4+ and CD8+ T cells in pretreatment samples: in CD8+ T cells, diversity index decreased in both groups after treatment, with higher difference in the VGCR group. CONCLUSION. Our study provides strong support that combined treatment induce humoral and cellular immune responses. We observed an activation of the immune system and a decrease in some immune checkpoints and MDSC. Finally, patients with a higher TCR-DI initially have a better clinical response and treatment induced oligoclonal activation of T cells. Citation Format: Laura Hato, Susana Inogés, Belén Pérez, Luis Mejías, Rodrigo Sánchez-Bayona, Marta Santisteban, Ascensión López. Immune response monitoring in breast cancer patients treated with neoadjuvant chemotherapy combined with dendritic cell vaccines [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-11.

Immune Checkpoint Blockade Enhances Mutated Calreticulin-Induced T Cell Immunity in Myeloproliferative Neoplasms

▪Somatic mutations in the calreticulin (CALR) gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. Each CALR mutation leads to a shift in the reading frame resulting in the formation of an altered protein with an identical 36-amino acid sequence in the C-terminus. We hypothesized that the recurrence and uniformity of the novel sequence would provide the mutated CALR an attractive candidate as a MPN-specific tumor antigen that might elicit anti-tumor immune responses. Therefore, we investigated the immunogenicity of the altered CALR by employing in silico peptide binding prediction algorithms to interrogate the binding affinities of mutated CALR peptides to human leukocyte antigens (HLA). These analyses indicated that the mutated epitopes can bind to class I and II HLA alleles with high affinity supporting the hypothesis that mutant CALR could be immunogenic in vivo . We then tested the immunogenicity of the overlapping peptides spanning the mutated region in vitro by utilizing intracellular staining (ICS) and Enzyme-Linked ImmunoSPOT (ELISPOT) assays. Naïve T cells derived from the peripheral blood mononuclear cells (PBMNCs) isolated from healthy donors displayed effector functions after priming with the mutated peptides but not with the corresponding wild type peptides . Incubation withthe mutated CALR peptides induced T cells proliferation, upregulation of CD137 (4-1BB) and production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Moreover, as suggested by in silico peptide-HLA binding predictions, both CD8+ and CD4+ T cell precursors displayed increased IFN-γ production in response to stimulation with mutated CALR peptides. There was an overall 4±3-fold increase in IFN-γ production by CD4+ T cells and a 2.5±0.5-fold increase by CD8+ T cells after stimulation with mutated peptides as compared to control self-antigens (n = 14 healthy donors out of which 11 showed CD4+ and 5 showed CD8+ T cell responses). These results demonstrate that mutated CALR elicits T cell responses in vitro supporting its potential immunogenic functions in vivo .We next investigated the presence of mutant CALR-specific T cell immunity in MPN patients with CALR mutations. PBMNCs from a total of 18 CALR-mutated MPN patients with ET (7), MF (5) or MF arising from ET (6) were evaluated. MPN T cells were then stimulated with the pooled mutant CALR peptides and T cell responses were assessed by ELISPOT. Out of 18 patients, increased IFN-γ production upon stimulation was observed in only 2 patients (one with ET and one with ET-MF). The less than robust MPN T cell responses suggested that immune suppressive mechanisms might be operating. To this end, we evaluated the expression of cell surface inhibitory receptors, programmed cell death (PD)-1 and the cytotoxic T-lymphocyte-associated antigen (CTLA)-4, by T cells from MPN patients. Both CD8+ and CD4+ T cells from MPN patients exhibited increased levels of PD-1 and CTLA-4 when compared to the healthy controls (HC). For instance, 9.3±8.2% of MPN patients CD4+ T cells expressed PD-1 (n = 12) as compared to 2.5±1.7% of HC CD4+ T cells (n=10) (p value = 0.0482). Similarly, there were twice as many PD-1 expressing CD8+ T cells in MPN patients as compared to HC. We next assessed MPN T cell responses against mutated peptides in the presence of neutralizing PD-1 or CTLA-4 antibodies. PD-1 inhibition restored T cell immunity in cultures derived from 3 MPN patients and CTLA-4 inhibition restored response in one patient (n=16). Of note, following the PD-1 or CTLA-4 inhibition, the number of IFN-γ producing cells and the amount of IFN-γ produced on a per-cell-basis increased upon stimulation with mutant-CALR peptides as compared to control peptides. These results indicate that the limited MPN T cell responses to CALR mutated peptides were due to T cell exhaustion caused, at least in part, by activation of the PD-1/CTLA-4 pathway.In summary, our studies establish mutated CALR as a MPN-specific tumor antigen thereby providing a rationale for the development of immunotherapies targeting mutated CALR in patients. Moreover, our data indicate that immune checkpoint signaling might be responsible for T cell exhaustion in some MPN patients thereby supporting the use of checkpoint blockade inhibitors for treatment of these patients. DisclosuresMascarenhas:Incyte: Other: Clinical Trial Steering Committee , Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding; Merck: Research Funding; Novartis: Other: DSMB member , Research Funding; Promedior: Research Funding. Bhardwaj:Merck & Co., Inc.: Membership on an entity's Board of Directors or advisory committees; Dendreon Corporation: Membership on an entity's Board of Directors or advisory committees; Dynavax Technologies Corporation: Consultancy; Crucell: Patents & Royalties; Neostem, Inc.: Patents & Royalties.

A systematic review and meta-analysis of enzyme-linked immunosorbent spot (ELISPOT) assay for BK polyomavirus immune response monitoring after kidney transplantation.

BK virus (BKV) infection after kidney transplantation can cause BKV nephropathy (BKVAN) resulting in graft dysfunction and allograft loss. The treatment for BKVAN is reduction of the immunosuppressive load which increases the risk of kidney transplant rejection. There is no biomarker to monitor BKV activity besides BK viral load. The value of the Enzyme-Linked Immunosorbent Spot (ELISPOT) assay as a tool to monitor the recipient's anti-BKV immune response after transplantation was investigated systematically. Electronic databases, including MEDLINE, Scopus, and the Cochrane Central Register of Controlled Trials were searched for studies of ELISPOT evaluating the immune response against BKV. BKV status was categorized as "active BKV infection" and as "resolving BKV infection". Random-effects model meta-analysis was performed to determine the diagnostic performance of the ELISPOT assay, after stratifying patients into groups based on positive and negative ELISPOT results. One-hundred twenty-seven articles were identified of which nine were included. Patients with negative ELISPOT had an increased risk of having active BKV replication (odds ratio of 71.9 (95%-CI 31.0-167.1). Pooled sensitivity was 0.95 (95%-CI 0.89-0.98) and specificity was 0.88 (95%-CI 0.78-0.94). The standardized mean difference of the number of IFN-γ producing cells between patients with active BKV infection compared with patients who had resolving BKV infection was -2.09 (95%-CI -2.50, -1.68). The ELISPOT assay is a useful tool for BKV risk assessment and in combination with BKV load may support clinicians in guiding immunosuppressive therapy in patients with BKV replication.

Clinical Application of iNKT Cell-mediated Anti-tumor Activity Against Lung Cancer and Head and Neck Cancer.

Invariant natural killer T (iNKT) cells produce copious amounts of cytokines in response to T-cell receptor (TCR) stimulation by recognizing antigens such as α-galactosylceramide (α-GalCer) presented on CD1d; thus, orchestrating other immune cells to fight against pathogen infection and tumors. Because of their ability to induce strong anti-tumor responses and the convenience of their invariant TCR activated by a synthetic ligand, α-GalCer, iNKT cells have been intensively studied for application in immunotherapeutic approaches to treat cancer patients in the clinic. Here, we summarize the clinical trials of iNKT cell based immunotherapy for non-small cell lung cancer, and head and neck cancer. Although solid tumors are thought to be refractory to immunotherapeutic approaches, our clinical trials showed that the intravenous injection of α-GalCer-pulsed antigen presenting cells (APCs) activated endogenous iNKT cells and iNKT cell dependent responses. Moreover, an increase in the number of IFN-γ producing cells in PBMCs was associated with prolonged survival. The marked infiltration of iNKT cells and the accumulation of conventional T cells in the tumor microenvironment were also observed after the administration of α-GalCer-pulsed APCs and/or ex vivo activated iNKT cells. In cases of advanced head and neck squamous cell carcinoma, the increased accumulation of iNKT cells in the tumor microenvironment was correlated with objective clinical responses. We will also discuss potential combination therapies of iNKT cell based immunotherapy to achieve enhanced anti-tumor activity and provide better treatment options for these patients.

Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease

The aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ production in chicken peripheral blood leucocytes.Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ− cells (αβ T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3− indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ−CD8α+ (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3+TCR1γδ−CD4+ (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

Increased interferon-mediated immunity following in vitro and in vivo Modafinil treatment on peripheral immune cells

The wake-promoting drug Modafinil has been used for treatment of sleep disorders, such as Narcolepsy, excessive daytime sleepiness and sleep apnea, due to its stimulant action. Despite the known effect of Modafinil on brain neurochemistry, particularly on brain dopamine system, recent evidence support an immunomodulatory role for Modafinil treatment in neuroinflammatory models. Here, we aimed to study the effects of in vitro and in vivo Modafinil treatment on activation, proliferation, cell viability, and cytokine production by immune cells in splenocytes culture from mice. The results show that in vitro treatment with Modafinil increased Interferon (IFN)-γ, Interleukin (IL)-2 and IL-17 production and CD25 expression by T cells. In turn, in vivo Modafinil treatment enhanced splenocyte production of IFN-γ, IL-6 and tumor necrosis factor (TNF), and increased the number of IFN-γ producing cells. Next, we addressed the translational value of the observed effects by testing PBMCs from Narcolepsy type 1 patients that underwent Modafinil treatment. We reported increased number of IFN-γ producing cells in PBMCs from Narcolepsy type 1 patients following continuous Modafinil treatment, corroborating our animal data. Taken together, our results show, for the first time, a pro-inflammatory action of Modafinil, particularly on IFN-mediated immunity, in mice and in patients with Narcolepsy type 1. The study suggests a novel effect of this drug treatment, which should be taken into consideration when given concomitantly with an ongoing inflammatory or autoimmune process.

Immunogenicity of twenty peptides representing epitopes of the hepatitis B core and surface antigens by IFN-γ response in chronic and resolved HBV.

BackgroundPatients with chronic hepatitis B virus infection (CHB) usually mount a modest T cell response against HBV epitopes. In order to determine immunogenic epitopes of HBV recognized by HBV-specific T cells, previous studies focused on previously confirmed HBV epitopes and assessed the T cell response by the number of HBV-specific T cells by IFN-γ ELISPOT.MethodsWe studied T cell functionality by combined in silico methods predicting HBV-specific epitopes and experimental investigations on the recognition of these epitopes. 30 chronic CHB patients and 10 patients with resolved HBV (RHB) were included in the study. We identified epitopes from the literature and by in silico analysis. These were evaluated for immunogenicity by use of synthetic peptides representing the epitopes through exposure to PBMCs from patients with CHB or RHB by IFN-γ ELISPOT. The number of IFN-γ producing cells (SFC), mean spot size (MSS) and stimulation index (SI) were recorded.ResultsThe frequency of HBV-specific T cells producing IFN-γ after stimulation with HBV epitopes was similar in CHB and RHB patients. CHB patients had a higher MSS SI than RHB patients. Patients not carrying the HLA-A2 genotype had higher SFC SI and MSS SI. Patients with HLA-A11 had higher MSS SI compared to non- HLA-A11 allele patients. HBeAg-positive patients had a lower MSS SI, and none of the HBeAg positive patients had the HLA-A11 genotype. We found 3 immunogenic epitopes not described previously.ConclusionIFN-γ ELISPOT-determined MSS is an efficient marker for T cell recognition of epitopes. This experimental measure showed the in silico analysis for epitope prediction to be a valuable tool in future studies on HLA genotypes and HBV epitopes. This way our study now points to previously unappreciated consequences of carrying the HLA-A11 allele in terms of stronger immunity to HBV.

Antigen targeting to dendritic cells allows the identification of a CD4 T-cell epitope within an immunodominant Trypanosoma cruzi antigen.

Targeting antigens to dendritic cells (DCs) by using hybrid monoclonal antibodies (mAbs) directed against DC receptors is known to improve activation and support long-lasting T cell responses. In the present work, we used the mAb αDEC205 fused to the Trypanosoma cruzi amastigote surface protein 2 (ASP-2) to identify a region of this protein recognized by specific T cells. The hybrid αDEC-ASP2 mAb was successfully generated and preserved its ability to bind the DEC205 receptor. Immunization of BALB/c mice with the recombinant mAb in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)) specifically enhanced the number of IFN-γ producing cells and CD4+ T cell proliferation when compared to mice immunized with a mAb without receptor affinity or with the non-targeted ASP-2 protein. The strong immune response induced in mice immunized with the hybrid αDEC-ASP2 mAb allowed us to identify an ASP-2-specific CD4+ T cell epitope recognized by the BALB/c MHCII haplotype. We conclude that targeting parasite antigens to DCs is a useful strategy to enhance T cell mediated immune responses facilitating the identification of new T-cell epitopes.

Major and minor histocompatibility antigens to NIMA: Prediction of a tolerogenic NIMA effect.

The immunologic effects of developmental exposure to non-inherited maternal antigens (NIMA) are heterogeneous, either tolerogenic or immunogenic. The role of minor histocompatibility antigens (MiHA) in NIMA effects is unknown. We have recently reported that the NIMA effect can be classified into two distinct reactivities, low and high responder, to NIMA in utero and during nursing depending on the degree of maternal microchimerism (MMc) and Foxp3 expression of peripheral blood CD4(+)CD25(+) cells after graft-versus-host disease (GVHD) induction. These reactivities were predictable before transplantation, using an MLR-ELISPOT (mixed lymphocyte reaction; enzyme-linked immunospot) assay by comparing the number of IFNγ-producing cells stimulated with NIMA. Moreover, this assay was also applicable in both major and minor NIMA-mismatched setting. These observations are clinically relevant and suggest that it is possible to predict the immunological tolerance to NIMA.

Targeting the Non-structural Protein 1 from Dengue Virus to a Dendritic Cell Population Confers Protective Immunity to Lethal Virus Challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4+ and CD8+ T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205+ DC population with poly (I:C) opens perspectives for dengue vaccine development.

Role of IRAK-M in Alcohol Induced Liver Injury

Increasing evidence suggests that innate immunity plays an important role in alcohol-induced liver injury and most studies have focused on positive regulation of innate immunity. The main objective of this study was to investigate the negative regulator of innate immunity, IL-1/Toll-like receptor (TLR) signaling pathways and interleukin receptor-associated kinase-M (IRAK-M) in alcoholic liver injury. We established an alcohol-induced liver injury model using wild type and IRAK-M deficient B6 mice and investigated the possible mechanisms. We found that in the absence of IRAK-M, liver damage by alcohol was worse with higher alanine transaminase (ALT), more immune cell infiltration and increased numbers of IFNγ producing cells. We also found enhanced phagocytic activity in CD68+ cells. Moreover, our results revealed altered gut bacteria after alcohol consumption and this was more striking in the absence of IRAK-M. Our study provides evidence that IRAK-M plays an important role in alcohol-induced liver injury and IRAK-M negatively regulates the innate and possibly the adaptive immune response in the liver reacting to acute insult by alcohol. In the absence of IRAK-M, the hosts developed worse liver injury, enhanced gut permeability and altered gut microbiota.

Development of a DNA-launched replicon as a vaccine for porcine reproductive and respiratory syndrome virus

Though a modified live attenuated vaccine (MLV) is available against porcine reproductive and respiratory syndrome virus (PRRSV), its limitations in protective efficacy, safety and few others warrant the development of newer vaccines. In this study, we have constructed a propagation-defective DNA-launched PRRSV replicon as a vaccine candidate and evaluated its immunogenicity and protective efficacy in a group of pigs along with MLV vaccinated group. Our data showed that prior to the intranasal challenge with a homologous strain of PRRSV, only MLV vaccinated pigs developed antibody response measured by ELISA and none of the pigs in any group developed PRRSV neutralizing antibodies in serum. The MLV vaccinated group also showed high PRRSV-specific INF-γ response, whereas the replicon-vaccinated pigs showed low but detectable INF-γ response. After 14 days post challenge, all groups showed similar PRRSV-specific serum neutralizing titers and were positive for PRRSV-specific ELISA antibody. In addition, the replicon-vaccinated group showed a significant reduction in viremia in comparison to the control group. In conclusion, vaccination with the PRRSV DNA-launched replicon decreased the viremia and viral load in bronchoalveolar lavage fluids of the PRRSV-challenged pigs and increased numbers of IFN-γ producing cells. Thus, the vaccine is partially protective and is a potential vaccine candidate for future with further improvement. The possible means of improvement is the expression of immunostimulatory genes by the replicon. We demonstrated the feasibility of this approach by expression of a foreign gene encoding firefly luciferase after transfection of cultured cells with the replicon plasmid DNA.

Azacitidine Induces A Shift In Th1/Th17 Ratio and FoxP3 Expression In Anti-CD3 Stimulated CD4+ T-Cells; Implications for the Treatment of Myelodysplastic Syndromes

AbstractAbstract 4026Aberrant DNA methylation and other epigenetic changes play a role in the development of myelodysplastic syndromes (MDS). Epigenetic drugs such as DNA methyltransferase inhibitors are therefore increasingly employed in MDS treatment regimens. Recent studies show that gene methylation processes also regulate T-cell function. Here we analyzed the in vitro effects of the DNA methyltransferase inhibitor '5-azacitidine (Aza) on CD4+ T-cell activation. We confirmed the previously described inhibition of proliferation and increased expression of FoxP3, the regulatory T-cell (Treg) marker, by anti-CD3 stimulated T-cells in the presence of 1mM Aza. Here we have sorted CD4+ T-cells isolated form healthy donor peripheral blood into CD25neg resting, CD25dim recently activated and CD25hi Treg cells. Aza facilitated the induction of CD25hiFoxP3+ T-cells from CD25neg (4.7% of vehicle treated cells versus 17.3% of Aza treated cells p=0.0007, n=9) and to a lesser extend from CD25dim (1.2% versus 8.6%, p=0.0015, n=7) CD4+ T-cells, while Aza had no effect on FoxP3 expression in CD25hi sorted cells, FoxP3 expression remained high. In addition, cytokine producing T-cells were enumerated after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin in the presence of Brefeldin A. Aza treatment increased the number of IFNγ producing cells in the total CD4+ population (19.1% versus 40.8%; p<0.0001, n=10) as well as among the CD25neg (5.7% vs 41.2%; p=0.001, n=8) and CD25dim CD4+ T-cell populations (28.4% versus 46.6%; p=0.06, n=7). TNFα producing cells were increased in the total CD4+ (36.4% versus 51.1%; p=0.011, n=9) and CD4posCD25dim (36.7% versus 52.3%; p=0.033, n=6) populations but not in the CD4posCD25neg cells (50.9% versus 51.1%; p=0.9, n=7). This increase in pro-inflammatory cytokine production indicates that Aza induces T-cell activation and that the increase in FoxP3 expression may reflect T-cell activation rather than an increase in bona fide Treg by Aza treatment. Indeed a proportion of the FoxP3+ cells was positive for TNFa or IFNg, suggesting that these are activated T-cells rather than Treg. However, the proportion of FoxP3+IFNγ- and FoxP3+TNFα- cells was significantly higher among Aza treated CD4+ cells (p=0.0037 and 0.0018 respectively, n=5), suggesting an increase in Treg as well. Functional assays to demonstrate that these FoxP3+ cells are indeed regulatory T-cells are currently being set up. Next to IFNγ and TNFα producing Th1 cells, the more recently described IL-17 committed Th17 cells have been described to play a role in low risk MDS. Furthermore, it has been shown that Treg can differentiate into IL-17 producing cells. We, therefore evaluated the effect of Aza on Th17 cells. Interestingly, in contrast to IFNγ and TNFα producing cells, the proportion of IL-17+IFNγ- Th17 cells among the total CD4+ population was reduced by Aza treatment (1.8% versus 1.1% p=0.035, n=10), leading to a significant increase in the Th1/Th17 ratio (14.0 versus 47.9, p=0.0005, n=10). In contrast, although the numbers were small, the proportion of Th17 cells was increased by Aza in the CD25neg population (0.08% versus 0.20%, p=0.028, n=8), suggesting that Aza may have differential effects on resting and recently activated T-cells. In conclusion, our data show that Aza increases the induction of FoxP3+ Treg and Th1 cells but inhibits IL-17 production, particularly by previously activated T-cells. Aza may therefore particularly be beneficial in pathogenic immune disorders characterized by increased Th17 numbers accompanied by reduced Treg frequencies, such as low-risk MDS. Disclosures:No relevant conflicts of interest to declare.

B7.1 and B7.2 are crucial for primary response to Mycobacterium tuberculosis infection but redundant for the induction of memory response (45.24)

Abstract Costimulation plays a crucial role in priming of naïve T cell to an effector cell type. In a recent study, we have shown that lack of B7.1 and B7.2 (DKO) causes the host to generate a dampened Th1 effector response, disabling it to contain the bacterial burden during chronic phase of infection. Since the development of T cell memory is connected to the effector T cell response, we sought to investigate how a defective primary effector response will affect the induction of the memory. Memory mice were generated by treating infected wildtype (WT) and DKO mice with antibiotics and resting them after the treatment. These memory mice along with age matched naïve controls were then challenged with low dose infection with M. tuberculosis. A significant decrease in bacterial burden was seen in both WT and DKO memory mice at 4 and 6 weeks post infection when compared to naive WT and DKO mice respectively. This decreased bacterial burden was accompanied with a faster Th1 effector response in both WT and DKO memory lungs as indicated by increased number of IFNγ producing cells in the lungs. These results indicate that despite its inability to generate a defective primary response the DKO host is able to mount a memory response against M.tuberculosis infection. Further studies are in progress to study the mechanism of memory induction in the absence of a successful primary response in the DKO mice. This study was supported by NIH Grant AI 49778, Potts Memorial Foundation Award and UMDNJ Foundation Award.

Galectin-9 Increases Tim-3+ Dendritic Cells and CD8+ T Cells and Enhances Antitumor Immunity via Galectin-9-Tim-3 Interactions

A Tim-3 ligand, galectin-9 (Gal-9), modulates various functions of innate and adaptive immune responses. In this study, we demonstrate that Gal-9 prolongs the survival of Meth-A tumor-bearing mice in a dose- and time-dependent manner. Although Gal-9 did not prolong the survival of tumor-bearing nude mice, transfer of naive spleen cells restored a prolonged Gal-9-induced survival in nude mice, indicating possible involvement of T cell-mediated immune responses in Gal-9-mediated antitumor activity. Gal-9 administration increased the number of IFN-gamma-producing Tim-3(+) CD8(+) T cells with enhanced granzyme B and perforin expression, although it induced CD4(+) T cell apoptosis. It simultaneously increased the number of Tim-3(+)CD86(+) mature dendritic cells (DCs) in vivo and in vitro. Coculture of CD8(+) T cells with DCs from Gal-9-treated mice increased the number of IFN-gamma producing cells and IFN-gamma production. Depletion of Tim-3(+) DCs from DCs of Gal-9-treated tumor-bearing mice decreased the number of IFN-gamma-producing CD8(+) T cells. Such DC activity was significantly abrogated by Tim-3-Ig, suggesting that Gal-9 potentiates CD8(+) T cell-mediated antitumor immunity via Gal-9-Tim-3 interactions between DCs and CD8(+) T cells.

In vitro evidence that commercial influenza vaccines are not similar in their ability to activate human T cell responses

We evaluated three commercial trivalent inactivated vaccines (TIVs) from the 2007–2008 season in terms of their ability to elicit in vitro T cell responses. T cell-mediated immunity may offer a more cross-reactive vaccine approach for the prevention of pandemic or epidemic influenza. Human cytotoxic T cell lines demonstrated differences in matrix protein 1 and nucleocapsid protein recognition of autologous target cells. Peripheral blood mononuclear cells stimulated with each of the TIVs showed statistically significant differences between the vaccines in the numbers of IFNγ producing cells activated. These data suggest that TIV vaccines are not similar in their ability to activate human T cell responses.

Decreased cytotoxic T cell activity generated by co-administration of PSA vaccine and CpG ODN is associated with increased tumor protection in a mouse model of prostate cancer

Immunization with an adenovirus-PSA (Ad5-PSA) vaccine alone strongly induces the expansion of CD8+ T cells with enhanced cytotoxic T lymphocyte (CTL) activity against the antigen-bearing tumor cells in vitro as well as in vivo in a mouse model of prostate cancer. However, in an attempt to enhance the anti-tumor immunity induced by the vaccine, co-administration of CpG oligodeoxynucleotides (CpG ODN) with Ad5-PSA vaccine dramatically reduces the immune responses measured by in vitro CTL activity and the number of IFN-γ producing cells. Surprisingly, in vivo experiments showed that mice immunized with the combined approach of Ad5-PSA and CpG had enhanced protection against the subsequent tumor challenge as compared to mice immunized with vaccine alone. These data demonstrate an unexpected dichotomous relationship between in vitro CTL activity and in vivo tumor protection suggesting that an alternative mechanism of tumor destruction was invoked after co-administration of the CpG ODN with the vaccine.

Tetrahydrocannabinol suppresses immune function and enhances HIV replication in the huPBL-SCID mouse

Epidemiologic studies identify marijuana as a potential cofactor in the development and progression of HIV infection. To evaluate this interaction we employed a hybrid model in which human peripheral blood leukocytes (PBL) were implanted into severe combined immunodeficient mice (huPBL-SCID) and infected with an HIV reporter construct in the presence or absence of tetrahydrocannabinol (THC) exposure. Administration of THC alone, in the absence of HIV, decreased CD4 counts and the CD4:CD8 ratio. Co-administration of THC and HIV did not reduce CD4 counts further, but significantly increased the percentage of HIV-infected PBL when compared to saline-treated animals (17 ± 4.6% vs. 7 ± 1.4%). Quantitative PCR confirmed a 50-fold increase in systemic viral load in THC-treated animals. The CCR5 and CXCR4 chemokine receptors function as coreceptors essential for HIV infection. Administration of THC for 5 days increased the percentage of PBL expressing CCR5 and, to a lesser extent, CXCR4. This effect was lost after 10 days of THC administration, but the number of HIV-infected cells had significantly increased by that time suggesting a role for early upregulation of these coreceptors in the pathogenic effect of THC. Finally, the impact of treatment on the number of human interferon-gamma (IFN-γ) producing cells was determined by ELISPOT. Both THC and HIV infection independently decreased the number of IFN-γ producing cells and co-administration produced additive effects. These results suggest that exposure to THC in vivo can suppress immune function, increase HIV coreceptor expression, and act as a cofactor to significantly enhance HIV replication.