Cell-dependent Immune Responses Research Articles

The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity

Tyrosine phosphorylation of signaling molecules that mediate B cell activation in response to various stimuli is tightly regulated by protein tyrosine phosphatases (PTPs). PTP1B is a ubiquitously expressed tyrosine phosphatase with well-characterized functions in metabolic signaling pathways. We show here that PTP1B negatively regulates CD40, B cell activating factor receptor (BAFF-R), and TLR4 signaling in B cells. Specifically, PTP1B counteracts p38 mitogen-activated protein kinase (MAPK) activation by directly dephosphorylating Tyr(182) of this kinase. Mice with a B cell-specific PTP1B deficiency show increased T cell-dependent immune responses and elevated total serum IgG. Furthermore, aged animals develop systemic autoimmunity with elevated serum anti-dsDNA, spontaneous germinal centers in the spleen, and deposition of IgG immune complexes and C3 in the kidney. In a clinical setting, we observed that B cells of rheumatoid arthritis patients have significantly reduced PTP1B expression. Our data suggest that PTP1B plays an important role in the control of B cell activation and the maintenance of immunological tolerance.

Nek2 is a novel regulator of B cell development and immunological response.

The serine/threonine kinase Nek2 is commonly found upregulated in a wide variety of neoplasms including diffuse large B cell lymphoma and multiple myeloma. High expression of Nek2 is implicated in the induction of chromosomal instability, promotion of cell proliferation, and drug resistance in tumor cells as well as a marker for poor clinical outcomes. Despite its well recorded involvement in chromosomal instability and neoplastic growth, little is known about the involvement of Nek2 in B cell development. Here we report the development of a transgenic mouse line with conditional expression of Nek2 in the B cell lineage and the effects it has on the development of B cells. Interestingly, we found that the overexpression of Nek2 does not induce spontaneous tumor formation within the transgenic mice up to 24 months after induction. Instead, overexpression of Nek2 in the B cell lineage affects the development of B cells by increasing the proportion of immature B cells in the bone marrow and decreasing B-1 B cells in peritoneal cavity. Furthermore, Nek2 transgenic mice develop spontaneous germinal centers and exhibit an enhanced T cell dependent immune response. Altogether, our data demonstrates a novel role for Nek2 in regulating B cell development and the immune response.

Distinct Cytokine Profiles in Patients with Oligoarticular Juvenile Idiopathic Arthritis after <i>in</i> <i>Vitro</i> Blockade of T Cells by Cyclosporine and Abatacept

Oligoarticular juvenile idiopathic arthritis (oJIA) is an antigen-driven and lymphocyte-mediated disorder affecting the adaptive immune system. Auto reactive T cells produce pro-inflammatory cytokines as IFN-γ and IL-17. Failure of regulatory T cells leads to decreased production of anti-inflammatory IL-10 and results in the loss of immune tolerance. Therapeutic strategies suppress T cell dependent immune responses and consequently inhibit the process of inflammation. The aim of the study was to investigate the effect of T cell suppression on the cytokine network in oJIA patients. Therefore we examined the cytokine concentration after in vitro inhibition of T cells by cyclosporine and abatacept in patients with persistent oJIA and healthy control subjects. This single center cohort study consisted of oJIA affected children and control subjects. Cytokine profiles from cell culture supernatants were examined with multiplex fluorescent bead immunoassay by flow cytometry. High amounts of IL-17 were only observed in the collective of oJIA patients after T cell stimulation. Cyclosporine suppresses its concentration effectively. IL-2 and IFN-γ are present in both groups. We found IL-6 and TNF-α in high concentrations after T cell activation. While TNF-α concentration is suppressed by both drugs, IL-6 concentration remains high in oJIA patients. Concentrations of IL-4 and IL-10 were not found to be influenced in status of activation or suppression. In conclusion, the results of the present study imply that IL-17 is the crucial T cell cytokine in oligoarticular JIA. Only cyclosporine could inhibit the secretion of IL-17 effectively. IL-2 and IFN-γ are not specific for oligoarticular JIA. Both cytokines are found as well in healthy control subjects after T cell stimulation. Relevant pro-inflammatory macrophage cytokines in oligoarticular JIA are TNF-α and IL-6. T cell suppression by cyclosporine and abatacept inhibits TNF-α but not IL-6 effectively. Production of anti-inflammatory cytokines is not influenced by T cell suppression.

Targeting Long-Lived Plasma Cells Is Essential For Treating Hemophilia A Inhibitors

The most critical and challenging problem for therapeutic treatment in hemophilia A patients is due to the formation of Inhibitory antibodies. It is hypothesized that long-lived plasma cells (LLPCs) play an important role in the persistent production of anti-FVIII antibodies in hemophilia A (HemA) inhibitor patients. The migration of plasma cells to the BM, where they become the LLPCs, is largely controlled by an interaction between the C-X-C type chemokine ligand 12 (CXCL12) produced by bone marrow (BM) stromal cells and its receptor CXC receptor4 (CXCR4; CD184) on plasma cells surface. Our previous data showed that administration of anti-murine CD20 (IgG2a) alone can deplete B cells significantly and reduce anti-FVIII inhibitor titers transiently in FVIII plasmid-treated HemA mice with pre-existing inhibitors, however, complete tolerance to FVIII was not achieved probably due to the persistence of LLPCs. We sought novel therapeutic strategies that target CXCL12/CXCR4 pathway to reduce/eliminate LLPCs and achieve the goal for long-term tolerance to FVIII in the HemA inhibitor mice. AMD3100, the CXCR4 antagonist, plus G-CSF inhibit the interaction of CXCL12 and CXCR4, thus facilitating the mobilization of stem cells and blocking the homing and retention of LLPCs. The combined treatment strategy used anti-CD20, G-CSF and AMD3100 to target B cells plus with the specific IL-2/IL-2mAb (JES6-1) complexes to target T cells for preventing both T and B cell-dependent anti-FVIII immune responses. Two groups of FVIII-primed inhibitor mice were treated with different combined immunomodulation regimens: (1) IL-2 complexes+AMD3100+G-CSF+anti-CD20, (2) AMD3100+G-CSF+anti-CD20. Control mouse groups were treated with each of the single regimens and FVIII only, or untreated as the naive control. All the treatments were administered one cycle per two weeks for 6 weeks and the therapeutic effects (FVIII activities) as well as immune responses (anti-FVIII inhibitors) were evaluated at different time points after treatment. Significant expansion of Treg cells reaching a 5∼7-fold increase on the peak days (day 3-7 after treatment) was observed in the IL-2/IL-2mAb complexes treated groups, whereas ∼95% of B cell populations were depleted in the anti-CD20 treated groups. In addition, administration of AMD3100 plus G-CSF significantly reduced circulating and bone marrow CXCR4+ plasma cells by flow cytometry analysis as well as decreased FVIII-specific plasma cells in bone marrow by ELISPOT assay. Except for the control groups, the two mouse groups treated with combined immunosuppressive regimens showed a significant reduction of inhibitory titers following the treatment. Long-term responses are being followed and second challenge with FVIII plasmid will be used to evaluate the induction of long term tolerance to FVIII. These combination regimens are highly promising in modulating/eliminating pre-existing anti-FVIII antibodies and inducing long-term tolerance in FVIII primed subjects. Disclosures:No relevant conflicts of interest to declare.

Selective Ablation of Lung Epithelial IKK2 Impairs Pulmonary Th17 Responses and Delays the Clearance of Pneumocystis

Pneumocystis is an atypical fungal pathogen that causes severe, often fatal pneumonia in immunocompromised patients. Healthy humans and animals also encounter this pathogen, but they generate a protective CD4(+) T cell-dependent immune response that clears the pathogen with little evidence of disease. Pneumocystis organisms attach tightly to respiratory epithelial cells, and in vitro studies have demonstrated that this interaction triggers NF-κB-dependent epithelial cell responses. However, the contribution of respiratory epithelial cells to the normal host response to Pneumocystis remains unknown. IκB kinase 2 (IKK2) is the upstream kinase that is critical for inducible NF-κB activation. To determine whether IKK2-dependent lung epithelial cell (LEC) responses contribute to the anti-Pneumocystis immune response in vivo, transgenic mice with LEC-specific deletion of IKK2 (IKK2(ΔLEC)) were generated. Compared to wild-type mice, IKK2(ΔLEC) mice exhibited a delayed onset of Th17 and B cell responses in the lung and delayed fungal clearance. Importantly, delayed Pneumocystis clearance in IKK2(ΔLEC) mice was associated with an exacerbated immune response, impaired pulmonary function, and altered lung histology. These data demonstrate that IKK2-dependent LEC responses are important regulators of pulmonary adaptive immune responses and are required for optimal host defense against Pneumocystis infection. LECs likely set the threshold for initiation of the pulmonary immune response and serve to prevent exacerbated lung inflammation by promoting the rapid control of respiratory fungal infection.

The impact of metabolic stress on anti-tumor immunity in laboratory mice

There is an underappreciated amount of physiological stress placed on laboratory mice including that due to standard, required housing at sub-thermoneutral temperature. This condition results in an increase in metabolism needed to produce heat necessary to maintain body temperature. Since body temperature maintenance is a high priority for survival, we hypothesized that metabolic energy may be allocated to body temperature maintenance at the expense of the anti-tumor immune response under standard cool housing temperatures. At ambient temperatures of 30-31°C, which is considered the thermoneutral temperature (TT) for mice, resting metabolic rate is sufficient to generate enough heat to maintain body temperature. However, mice are kept at a cooler standard temperature (ST; 21-23°C). Our data reveals increased heat-seeking behavior in tumor-bearing mice housed at ST indicating a physiological drive for additional ambient warmth, likely needed to lessen the energetic burden of maintaining body temperature. We found significantly delayed tumor growth rates and a reduction in metastasis in mice housed at TT compared to those at ST. We investigated immunodeficient mice and mice depleted for CD8+ T cells and found no differences in tumor growth between mice housed at ST and TT suggesting a role for the T cell dependent immune response. Because the activation of CD8+ T cells is an energetically demanding process we predicted the function of these cells would be diminished in animals at ST since more metabolic resources may be diverted to heat production. According to this hypothesis we saw an increase in glucose uptake in the lymph nodes of tumor-bearing mice as well as an increase in Glut-1 expression on splenic CD8+ T cells in mice maintained at TT suggesting increased metabolic activity in these cell populations. Our studies also revealed more antigen specific CD8+ T cells within the lymph node and tumor microenvironment of animals housed at TT compared to those housed at ST. Finally, our data has also shown a significantly increased number of immunosuppressive cell subsets including Gr-1+CD11b+ myeloid derived suppressor cells and Foxp3+ cells in mice maintained at ST compared to those at TT. These data demonstrate that tumor growth and anti-tumor immune control in laboratory mice is highly influenced by cold stress. We hypothesize that the amount of energy available to T cells under standard cool housing conditions is reduced. Since metabolism and its role in T cell activation is increasingly modeled in laboratory mice, it is essential to take into account the impact that housing temperature has on anti-tumor immune activity.

Hepatitis Virus Infection Affects DNA Methylation in Mice With Humanized Livers

Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes.

T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation

Protein therapeutics hold a prominent and rapidly expanding place among medicinal products. Purified blood products, recombinant cytokines, growth factors, enzyme replacement factors, monoclonal antibodies, fusion proteins, and chimeric fusion proteins are all examples of therapeutic proteins that have been developed in the past few decades and approved for use in the treatment of human disease. Despite early belief that the fully human nature of these proteins would represent a significant advantage, adverse effects associated with immune responses to some biologic therapies have become a topic of some concern. As a result, drug developers are devising strategies to assess immune responses to protein therapeutics during both the preclinical and the clinical phases of development. While there are many factors that contribute to protein immunogenicity, T cell- (thymus-) dependent (Td) responses appear to play a critical role in the development of antibody responses to biologic therapeutics. A range of methodologies to predict and measure Td immune responses to protein drugs has been developed. This review will focus on the Td contribution to immunogenicity, summarizing current approaches for the prediction and measurement of T cell-dependent immune responses to protein biologics, discussing the advantages and limitations of these technologies, and suggesting a practical approach for assessing and mitigating Td immunogenicity.

The Chronicity of Tonsillitis Is Significantly Correlated with an Increase in an LTi Cell Portion

The current study explored the relationship between lymphoid tissue inducer (LTi) cells and patients' clinical and immunological status. LTi cells are critical for lymphoid tissue development and maintenance of CD4 T cell-dependent immune responses. The percentage of CD117+CD3-CD56-CD127+ RORγ+ LTi cells isolated from human tonsils was determined and correlated with changes in other immune subsets and clinical factors. We found that the portion of LTi and CD4 T cells was significantly increased in chronic tonsillitis compared to non-inflamed tonsils. Additionally, the expression of OX40 by memory CD4 T cells and OX40 ligand (OX40L) and interleukin (IL)-22 by LTi cells was higher in chronically inflamed tonsils. The treatment for tonsillitis with ibuprofen did not alter LTi cell viability and the expression of OX40L and IL-22. These results demonstrate that during chronic inflammation, LTi cells are increased and express higher levels of OX40L and IL-22, and this is correlated with an increase in memory CD4 T cells.

Pathoetiology of multiple sclerosis: are we barking up the wrong tree?

Despite a century of intensive investigation, the underlying cause of multiple sclerosis has eluded us. It is clear that there exists a prominent progressive degenerative phenotype together with an important autoimmune inflammatory component, and careful histopathological examination always shows, to a greater or lesser degree, concomitant degeneration/demyelination and adaptive T cell-dependent immune responses. Given this picture, it is difficult, if not impossible, to definitively say whether degeneration or autoimmunity is the initiator of the disease. In this review, I put forward the evidence for and against both models and speculate that, in contrast to the accepted view, it is equally likely that multiple sclerosis may be a degenerative disease that secondarily elicits an autoimmune response, and suggest how this might influence therapeutic approaches.

Influence of initial vaccination with 13-valent pneumococcal conjugate vaccine or 23-valent pneumococcal polysaccharide vaccine on anti-pneumococcal responses following subsequent pneumococcal vaccination in adults 50 years and older

BackgroundUnlike free polysaccharide vaccines, pneumococcal polysaccharide conjugate vaccines (PCVs) induce a T cell-dependent immune response and have the potential to provide an extended duration of protection with repeated vaccinations. MethodsThis was an extension of a previous study in pneumococcal vaccine-naïve adults aged 50–64 years in which adults 60–64 years of age were given 13-valent PCV (PCV13) or 23-valent pneumococcal polysaccharide vaccine (PPSV23) and adults aged 50–59 were given PCV13. In this follow up study conducted about 4 years later, the 60–64 year olds initially given PCV13 received PCV13 or PPSV23, and those initially given PPSV23 received another PPSV23. All adults aged 50–59 years were re-vaccinated with PCV13. Anti-pneumococcal opsonophagocytic activity (OPA) titers were measured before and 1 month after vaccination. ResultsA second PCV13 given about 4 years after a first vaccination induced OPA titers that were significantly higher than those following the initial vaccination for 7 of 13 serotypes in the older group, and 6 of 13 serotypes in the younger group, and responses to the remaining serotypes were largely non-inferior. In contrast, OPA titers following revaccination with PPSV23 were statistically significantly lower for 9 of the 13 serotypes, and non-inferior for the remaining serotypes, when compared to the responses to the first PPSV23. OPA titers in the older adults who received PPSV23 after initial PCV13 were significantly higher than those following a first PPSV23 for 10 of the 13 serotypes. ConclusionIn adults 50 to 64 years of age, initial vaccination with PCV13 establishes an immune state that results in recall anti-pneumococcal responses upon subsequent vaccination with either conjugated or free polysaccharide vaccine. In contrast, initial vaccination with PPSV23 results in an immune state in which subsequent PPSV23 administration yields generally lower responses compared with the initial responses.

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections. Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed (1,2). This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a naïve mouse and DTH-like swelling is measured after 18-24 hr (3). The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens. The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts (2,4-14). The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function. A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity(15,16) and also in tumor immunology (17).

Immunotherapy of disseminated intraperitoneal pancreatic cancer with non-replicating Toxoplasma gondii (P2104)

Abstract Pancreatic cancer is one of the most devastating and deadly cancers with an overall 5-year survival rate of 5%. Given existing therapies offer limited benefit, there is a strong need for innovative treatments that boost the host immune response to cancer. Toxoplasma gondii is an obligate intracellular parasite that potently stimulates the immune system and elicits CD8 T cell dependent immune responses. Utilizing an intraperitoneal dissemination model of pancreatic cancer, we hypothesize a non-replicating strain of T. gondii (CPS) can alter the tumor microenvironment and elicit strong antitumor responses. Intraperitoneal PAN02 tumors were established and treated with CPS seven days later. CPS treatment significantly increased survival and some treated mice survived long term (>1 year). Treatment efficacy was completely dependent on production of IL-12 and IFN-γ. Within the peritoneal exudate, there is a significant decrease in B cells and classical macrophages and a significant increase in neutrophils, CD4, and CD8 T cells compared to untreated tumors. Therapeutic benefit was dependent on the presence of CD8 T cells whereas CD4 T cells did not significantly contribute to the survival advantage in treated mice. CPS treatment increased the frequency of pancreatic tumor-specific IFN-γ producing CD8 T cells. Further investigation is needed to characterize innate immune mechanisms supporting tumor therapy as well as the potential induction of immune memory.

T cell dependent induction of novel mast cell-lineage cells in spleen (P6006)

Abstract Mast cells and basophil are known to exert a variety of functions in normal as well as pathological immune responses. Although mutual dependencies between mast cell lineage cells (MCs) and T cells have been recently well documented, how antigen-specific T cell responses leads to differentiate MCs and to MCs-dependent immune responses is not fully understood. When we attempted to generate antigen-specific T cell lines from OVA and KLH immunized mice by repeatedly stimulating their spleen cells or lymph node (LN) cells in vitro, we observed dominant expansion of FcϵR1 and c-kit positive cells in spleen cell culture, not in LN cell culture. Although this surface phenotype has been classified as that of MC, toluidine blue staining and electron microscopic analysis demonstrated that granules found in these cells appeared more like those seen in classic basophils rather than MCs. The functions of these cells were equivalent to those of classical MCs or basophils such as β-hexomidase release in vitro and PCA reaction in vivo. Consistently, expression pattern of MCPs revealed mixed phenotypes of MCs and basophils. These cells produced IFN-γ when they were co-cultured with antigen specific T cells in the presence of relevant antigen, which occurred in mast cell-T cell contact manner. These results suggested the existence of unique mast cell lineage in spleen contributing T cell dependent immune responses. Roles of these cells in vivo are currently under investigation.

Costimulatory Molecules on Immunogenic Versus Tolerogenic Human Dendritic Cells

Dendritic cells (DC) are sentinels of immunity, essential for homeostasis of T cell-dependent immune responses. Both functions of DC, initiation of antigen-specific T cell immunity and maintenance of tissue-specific tolerance originate from distinct stages of differentiation, immunogenic versus tolerogenic. Dependent on local micro milieu and inflammatory stimuli, tissue resident immature DC with functional plasticity differentiate into tolerogenic or immunogenic DC with stable phenotypes. They efficiently link innate and adaptive immunity and are ideally positioned to modify T cell-mediated immune responses. Since the T cell stimulatory properties of DC are significantly influenced by their expression of signal II ligands, it is critical to understand the impact of distinct costimulatory pathways on DC function. This review gives an overview of functional different human DC subsets with unique profiles of costimulatory molecules and outlines how different costimulatory pathways together with the immunosuppressive cytokine IL-10 bias immunogenic versus tolerogenic DC functions. Furthermore, we exemplarily describe protocols for the generation of two well-defined monocyte-derived DC subsets for their clinical use, immunogenic versus tolerogenic.

Therapeutic Potential of Tolerogenic Dendritic Cells in IBD: From Animal Models to Clinical Application

The gut mucosa undergoes continuous antigenic exposure from food antigens, commensal flora derived ligands, and pathogens. This constant stimulation results in controlled inflammatory responses that are effectively suppressed by multiple factors. This tight regulation, necessary to maintain intestinal homeostasis, is affected during inflammatory bowel diseases (IBD) resulting in altered immune responses to harmless microorganisms. Dendritic cells (DCs) are sentinels of immunity, located in peripheral and lymphoid tissues, which are essential for homeostasis of T cell-dependent immune responses. The expression of a particular set of pathogen recognition receptors allows DCs to initiate immune responses. However, in the absence of danger signals, different DC subsets can induce active tolerance by inducing regulatory T cells (Treg), inhibiting inflammatory T helper cell responses, or both. Interestingly, several protocols to generate clinical grade tolerogenic DC (tol-DCs) in vitro have been described, opening the possibility to restore the intestinal homeostasis to bacterial flora by cellular therapy. In this review, we discuss different DC subsets and their role in IBD. Additionally, we will review preclinical studies performed in animal models while describing recent characterization of tol-DCs from Crohn's disease patients for clinical application.

SLC gene-modified dendritic cells mediate T cell-dependent anti-gastric cancer immune responses in vitro

Dendritic cells (DCs) are potent professional antigen-presenting cells (APCs) with the ability to prime naïve T cells, and play an important role in the initiation and regulation of immune responses. In this study, we constructed a recombinant adenovirus carrying the SLC gene (Ad-SLC), and detected the biological effects of Ad-SLC-modified DCs as an adjuvant for the initiation of gastric cancer immune responses. Human DCs were transfected with Ad-SLC and the recombinant adenovirus carrying the β-galactosidase gene, Ad-LacZ, respectively. Modified DCs were pulsed with the cell lysate antigen of SGC-7901 cells (a type of gastric cancer cell line) and co-cultured with autologous T cells. The T cells were harvested and incubated with SGC-7901 cells and the cytotoxic function of the T cells was detected. Based on the data, the expression of mature DC phenotypes CD83 and CCR7 was upregulated after transfection with Ad-SLC and the chemotaxis function of DCs was augmented after transfection with Ad-SLC. Moreover, the expression of RANTES in DCs was upregulated by Ad-SLC transfection, while expression levels of IL-12p70 and IL-10 were not significantly altered. When co-cultured with autologous T cells, DCs modified with the SLC gene and pulsed with SGC-7901 cell lysates significantly promoted the proliferation of autologous T cells and induced Th1 differentiation, and displayed a strong cytotoxicity to SGC-7901 cells. In conclusion, Ad-SLC promoted DC maturation, enhancing the ability of DCs for T-cell chemotaxis and T-cell stimulation, and induced specific anti-gastric cancer cellular immunity. Recombinant Ad-SLC-modified DCs may be used as an adjuvant to induce an effective anti-gastric cancer immune response.

Immunomodulation of Both Donors and Recipients with Atorvastatin As a Strategy for the Prevention of Acute Graft-Versus-Host Disease (aGVHD): Results of Two Parallel Prospective Trials in Recipients of Matched Sibling Allogeneic Hematopoietic Cell Transplantation (alloHCT)

AbstractAbstract 1942 Introduction:Acute GVHD is a leading cause of morbidity and mortality following alloHCT. Atorvastatin (a statin drug) is a potent immunomodulatory agent capable of suppressing T cell–dependent immune responses that holds promise as a novel and safe agent for the prophylaxis of aGVHD. In murine models atorvastatin administration to both donor and recipient mice (compared to prophylaxis in either donors or recipients alone), prevented aGVHD by inhibiting donor T-cell proliferation, inducing T-helper 2 polarization, and by inhibiting recipient antigen presenting cell function (Zeiser et al. Blood, 2007). Several retrospective studies also suggest statins as potential prophylactic agents for aGVHD. Methods:We report here the results of two parallel, ongoing, prospective, phase II trials (NCT01175148 &NCT01491958) evaluating the safety and efficacy of a ‘two-pronged' strategy of atorvastatin administration as aGVHD prophylaxis, to both adult (age ≥18yrs) donors and recipients of matched sibling alloHCT. The approach was identical in both protocols. Subjects with a history of atorvastatin allergy/intolerance were not eligible. As GVHD prophylaxis, atorvastatin at 40mg/day orally was administered to sibling donors, starting 14–28 days before the anticipated 1st day of stem cell collection. In alloHCT recipients GVHD prophylaxis consisted of atorvastatin (40mg/day) administered from day -14 to day +180 (or until stopping immunosuppression, toxicity, development of grade [Gr] II-IV aGVHD, or severe chronic GVHD [cGVHD]). In addition all recipients received standard GVHD prophylaxis with tacrolimus and methotrexate. Ex vivo CD34+ cell selection or in vivo T-cell depletion was not permitted. Primary outcomes were rate of Gr II-IV aGVHD at day +100 and safety of atorvastatin administration to alloHCT donor/recipient pairs. Both trials tested the null hypothesis H0: p≥35%, vs. the alternate H1: p≤15%, where p is probability of Gr II-IV aGVHD at day 100. Secondary outcomes included rates of late-onset aGVHD, cGVHD, relapse, progression-free survival (PFS), and overall survival (OS). Results:Between September 2010 and June 2012, 34 donor/recipient pairs were enrolled at WVU (n=24) and OSU (n=10). Median donor age was 50.5 yrs (range 24–69). Median duration of atorvastatin prophylaxis in donors was 15 days (range 7–26). No atorvastatin related Gr 3–4 toxicities were seen in the healthy donors. Table 1 shows baseline patient (pt) characteristics. At transplantation 16 pts (47%) had high-risk disease, 12 (35%) were chemorefractory, 11 (32%) had HCT-CI of ≥3, 14 (41%) were female to male allografts, and 12 (35%) pts had ABO mismatched transplants. Median follow up of survivors is 158 days (range 31 – 658). Atorvastatin was well tolerated with no Gr 2–4 adverse events. All pts engrafted. The median time to ANC ≥500/μL was 18 days (range 5–25) and to platelets ≥20k/μL was 15 days (range 11–51). The median day 100 chimerism was 85.5% (55–100%) for T-cells and 100% (26–100%) for myeloid cells. Respective numbers at day 180 are 99.5% and 100%. Among 34 evaluable pts, the cumulative incidences (CI) of Gr II-IV and Gr III–IV aGVHD at day 100 were 6.0 ± 4.2% and 0% respectively. Respective CI rates at day 180 are 16.7 ± 8.3% and 10.2 ± 7%. CI of mild/moderate and severe cGVHD at 1 year are 15.8 ± 8.9% and 18.3 ± 10.2%, respectively. Infectious complications were infrequent, with no fungal infections or EBV reactivations and few CMV reactivations (n=3). Non-relapse mortality was 0% at day 100 and 4.5% at day 180. CI of relapse at day 180 was 27.6 ± 9.2%. One year PFS and OS estimates are 54% and 66%, respectively. Conclusions:A two-pronged immunomodulatory strategy of atorvastatin administration to alloHCT donors/recipients appears to be a feasible, safe and potentially effective modality for aGVHD prophylaxis. Final results of NCT01175148 & updated results of NCT01491958 will be presented at the meeting.Table 1CharacteristicsPatients (N=34)Male gender21Median age (range)51.5 (22–67)Previously used statins5Median days on statin (range)128.5 (31–199)Median KPS (range)90 (70–100)Median HCT-CI (range)2 (0–5)CIBMTR Disease RiskLow9Intermediate9High16DiagnosisALL2AML9CML2MDS/CMML7CLL/SLL2NHL/Hodgkin8Others4Chemorefractory12Prior autograft6ConditioningRIC20Myeloablative14Peripheral blood graft34ABO mismatched12Median infused CD34 cells/kg4.6 × 10*6Median infused CD3 cells/kg31.9 × 10*7 Disclosures:Hamadani:Celgene Corp: Speakers Bureau; American Cancer Society 116837-IRG-09-061-01: Research Funding; ASBMT & Millennium New Investigator Award: Research Funding; Conquer Cancer Foundation of ASCO: Research Funding. Off Label Use: Off Label use of atorvastatin for GVHD prophylaxis. Efebera:(NIA) K12 CA1333250: Research Funding; Leukemia and Lymphoma Society: Research Funding.

B and T cells collaborate in antiviral responses via IL-6, IL-21, and transcriptional activator and coactivator, Oct2 and OBF-1

A strong humoral response to infection requires the collaboration of several hematopoietic cell types that communicate via antigen presentation, surface coreceptors and their ligands, and secreted factors. The proinflammatory cytokine IL-6 has been shown to promote the differentiation of activated CD4(+) T cells into T follicular helper cells (T(FH) cells) during an immune response. T(FH) cells collaborate with B cells in the formation of germinal centers (GCs) during T cell-dependent antibody responses, in part through secretion of critical cytokines such as IL-21. In this study, we demonstrate that loss of either IL-6 or IL-21 has marginal effects on the generation of T(FH) cells and on the formation of GCs during the response to acute viral infection. However, mice lacking both IL-6 and IL-21 were unable to generate a robust T(FH) cell-dependent immune response. We found that IL-6 production in follicular B cells in the draining lymph node was an important early event during the antiviral response and that B cell-derived IL-6 was necessary and sufficient to induce IL-21 from CD4(+) T cells in vitro and to support T(FH) cell development in vivo. Finally, the transcriptional activator Oct2 and its cofactor OBF-1 were identified as regulators of Il6 expression in B cells.

Primary sterile necrotic cells fail to cross-prime CD8+ T cells

Necrotic cells are known to activate the innate immune system and trigger inflammation by releasing damage associated molecular patterns (DAMPs). However, how necrotic cells influence the induction of antigen-specific CD8+ T cell-mediated adaptive immune responses under sterile conditions, in the absence of pathogen associated molecular patterns (PAMPs), remains poorly understood. Here, we examined antigen-specific CD8+ T-cell responses to primary sterile necrotic tumor cells both in vitro and in vivo. We found that primary necrotic cells alone fail to generate CD8+ T cell-dependent immune responses toward cell-associated antigens. We show that necrotic cells trigger CD8+ T-cell immunity only in the presence of PAMPs or analogs, such as p(dI-dC) and/or unmethylated CpG DNA. The electroporation of tumor cells with these PAMPs prior to necrosis induction triggered antigen-specific CD8+ T-cell responses through a TLR9/MyD88-dependent pathway. In addition, we found that necrotic cells contain factors that can block the cross-priming of CD8+ T cells even under non-sterile conditions and can serve as a possible mechanism of immunosuppression. These results suggest that antigen-specific CD8+ T-cell responses to primary necrotic tumor cells can be induced in the presence of PAMPs and thus have a substantial impact on the development of antitumor vaccination strategies.