Augmentation Of Immune Response Research Articles

Overexpression of Bovine FcRn in Mice Enhances T-Dependent Immune Responses by Amplifying T Helper Cell Frequency and Germinal Center Enlargement in the Spleen.

The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress the bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In the current study, we analyzed the localization of the bFcRn in secondary lymphoid organs, and focused to demonstrate the in vivo impact of its overexpression in the spleen on the course of antibody production. bFcRn was highly expressed by red pulp macrophages and marginal zone macrophages in the spleen and by subcapsular sinus macrophages and macrophage-like cells in the interfollicular areas in the lymph node cortex. We also demonstrated that splenic dendritic cells of Tg mice express bFcRn and intraperitoneal immunization of these mice with T-dependent antigens led to more than threefold increase in the number of antigen-specific activated T helper cells with increased size and numbers of germinal centers compared to wild-type controls. bFcRn expression in splenic B cells was also detected and that may also contribute to the enhanced B cell activation. Finally, we demonstrated that these Tg mice developed efficient immune response against very low dose of antigen, reflecting another important practical benefit of these Tg mice.

Long-Term Lung Transplantation in Nonhuman Primates

Despite advances in surgical technique and clinical care, lung transplantation still remains a short-term solution for the treatment of end-stage lung disease. To date, there has been limited experience in experimental lung transplantation using nonhuman primate models. Therefore, we have endeavored to develop a long-term, nonhuman primate model of orthotopic lung transplantation for the ultimate purpose of designing protocols to induce tolerance of lung grafts. Here, we report our initial results in developing this model and our observation that the nonhuman primate lung is particularly prone to rejection. This propensity toward rejection may be a consequence of 1) upregulated nonspecific inflammation, and 2) a larger number of pre-existing alloreactive memory T cells, leading to augmented deleterious immune responses. Our data show that triple-drug immunosuppression mimicking clinical practice is not sufficient to prevent acute rejection in nonhuman primate lung transplantation. The addition of horse-derived anti-thymocyte globulin and a monoclonal antibody to the IL-6 receptor allowed six out of six lung recipients to be free of rejection for over 120 days.

Expression of IFNgR on tumor cells impact response to anti-CTLA-4 (TUM2P.1034)

Abstract Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, Ipilimumab (BMS), has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of Ipilimumab for the treatment of metastatic melanoma. Our published data indicate that anti-CTLA-4 treatment resulted in marked increase of IFN-gamma (IFNg) production by CD4 T cells expressing inducible costimulator (ICOS). Studies in mice have demonstrated the importance of IFNg for effective anti-tumor responses. We hypothesized that tumor cell expression of IFNg-receptor (IFNg-R) would be necessary for tumor regression mediated by anti-CTLA-4. To test our hypothesis, we conducted both in vitro and in vivo experiments with tumor cell lines containing knockdown of IFNg-R. In the presence of IFNg, proliferation of tumor cells was inhibited; however, tumor cells lacking IFNgR were resistant to IFNg and were able to proliferate. In addition, mice inoculated with tumor cells lacking IFNgR were treated with anti-CTLA-4 and found to have impaired tumor rejection and higher mortality as compared to mice bearing tumor cells with intact IFNgR. Our data strongly suggest that intact IFNg-R expression and signaling on tumor cells is important for anti-CTLA-4-mediated anti-tumor responses. Our data point to a potential mechanistic pathway to explain why some patients may respond to therapy while others do not and we’re evaluating tumor samples from patients treated with anti-CTLA-4 to test this concept.

Immunity to the vacuolar ATPase complex accessory unit ATP6S1 in patients with malignant melanoma.

The augmentation of high-titer antibodies to ATP6S1 is associated with favorable clinical outcomes in patients who received vaccination with autologous, irradiated tumor cells engineered to secrete GM-CSF and allogeneic bone marrow transplantation. Cellular immune responses to ATP6S1 are unknown. To define its role as an immune target, examination of cellular responses to ATP6S1 and immunity related to current therapies such as checkpoint blockade is needed. We used an overlapping peptide library representing the full-length ATP6S1 protein to screen for cellular responses from the peripheral blood of patients with stage III and IV melanoma. Reactive peptide pools were used to determine the individual peptide activity and epitopes. Recombinant ATP6S1 protein was used in an ELISA to assess potential correlation with humoral immune responses and changes in immunity related to CTLA-4 blockade with ipilimumab in these patients. We observed a broad array of CD4(+) and CD8(+) cellular responses against ATP6S1, including the identification of several MHC class I and II ATP6S1 epitopes. The generation of specific CD4(+) and cytotoxic T cells revealed potent functional capability elicited by ipilimumab treatment in patients with metastatic melanoma, which revealed potent functional capability, including cytokine production, proliferation responsiveness to melanoma cell lines, and tumor-cell killing. Furthermore, the augmented humoral immune responses to ATP6S1 as a function of ipilimumab treatment were associated with beneficial clinical outcomes. These results support the continued development of ATP6S1 as a biomarker and therapeutic target.

Enhanced Humoral Immunity in Mice Lacking CB1 and CB2 Receptors (Cnr1 −/− /Cnr2 −/− Mice) is not Due to Increased Splenic Noradrenergic Neuronal Activity

Peripheral sympathetic noradrenergic neurons originating in the celiac mesenteric plexus have axons that terminate in close proximity to antibody-producing B cells in the spleen. Norepinephrine (NE) released from these neurons is reported to augment antibody production in response to an immune challenge via an action at the β2-adrenergic receptor (β2AR). Cannabinoids are immunosuppressive, and mice lacking CB1 and CB2 receptors (Cnr1(-/-)/Cnr2(-/-) mice) have augmented cell-mediated immune responses. The purpose of this study was to determine if Cnr1(-/-)/Cnr2(-/-) mice also exhibit enhanced humoral immunity and if that is associated with corresponding changes in noradrenergic neurons terminating in the spleen. The results reveal that IgM and IgG are enhanced in Cnr1(-/-)/Cnr2(-/-) mice as compared to WT both in immunologically naïve and lipopolysaccharide (LPS)-treated mice. While the elevated antibody production was correlated with increased expression of β2AR on splenic B cells and increased splenic capsule NE concentrations, the activity of noradrenergic neurons was suppressed in spleens from Cnr1(-/-)/Cnr2(-/-) mice as compared with WT controls. Together, these results suggest that Cnr1(-/-)/Cnr2(-/-) mice exhibit enhanced NE vesicular storage in axon terminals in these neurons, which might limit the NE available to bind β2AR on target cells, such as B cells. The results also demonstrate that enhanced antibody responses in the absence of CB1 and CB2 receptors are not due to increased sympathetic noradrenergic neuronal activity in the spleen.

Heteroglucan-dendrimer glycoconjugate: a modulated construct with augmented immune responses and signaling phenomena

BackgroundNewer strategies for augmenting immune responses of pharmacologically active glucans may serve to improve the medicinal potential of these biomolecules. With this aim, the present work was focused on generating targeted high molecular size glucan particles with magnified immune response activity. MethodsHeteroglucans were conjugated with PAMAM dendrimers using a Schiff base reductive amination reaction to generate a polytethered molecule with multiple glucan motifs. The modulated construct was characterized by FTIR, TEM, 1H NMR and dynamic light scattering (DLS) methods. Effects of conjugated glucans were examined in RAW 264.7 macrophage cells as well as in S-180 murine tumor models. ResultsDendrimer-conjugated glucans were found to exhibit a two-fold increase in immune stimulation in comparison to unconjugated glucans. This may be corroborated by the predominant enhancement in immunological functions such as nitric oxide production, ROS generation and immune directed tumor inhibition in murine models. Immune cell surface markers (CD4, CD8, CD19, MHC-II) and cytokine levels were also found to be highly up-regulated in the splenocytes of mice subjected to particulate glucan administration. Our study also demonstrated that conjugated glucan treatment to RAW 264.7 cells strongly enhanced the phosphorylation of two downstream signalling molecules of the mitogen activated protein kinase (MAPKs) family: p38 and MEK1/2 relative to single glucans thereby relating molecular mechanisms with enhanced immune stimulation. Conclusions and general significanceThe results obtained thus support that particulate format of soluble heteroglucan will thereby improve its functionality and identify leads in therapeutic competence.

Augmented Replicative Capacity of the Boosting Antigen Improves the Protective Efficacy of Heterologous Prime-Boost Vaccine Regimens

Prime-boost immunization regimens have proven efficacious at generating robust immune responses. However, whether the level of replication of the boosting antigen impacts the magnitude and protective efficacy of vaccine-elicited immune responses remains unclear. To evaluate this, we primed mice with replication-defective adenovirus vectors expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP), followed by boosting with either LCMV Armstrong, which is rapidly controlled, or LCMV CL-13, which leads to a more prolonged exposure to the boosting antigen. Although priming of naive mice with LCMV CL-13 normally results in T cell exhaustion and establishment of chronic infection, boosting with CL-13 resulted in potent recall CD8 T cell responses that were greater than those following boosting with LCMV Armstrong. Furthermore, following the CL-13 boost, a greater number of anamnestic CD8 T cells localized to the lymph nodes, exhibited granzyme B expression, and conferred improved protection against Listeria and vaccinia virus challenges compared with the Armstrong boost. Overall, our findings suggest that the replicative capacity of the boosting antigen influences the protective efficacy afforded by prime-boost vaccine regimens. These findings are relevant for optimizing vaccine candidates and suggest a benefit of robustly replicating vaccine vectors. The development of optimal prime-boost vaccine regimens is a high priority for the vaccine development field. In this study, we compared two boosting antigens with different replicative capacities. Boosting with a more highly replicative vector resulted in augmented immune responses and improved protective efficacy.

Inflammatory responses of older Firefighters to intermittent exercise in the heat

Repeated strenuous work in the heat may predispose firefighters to augmented immune responses and inflammation. This study examined the immune responses in 12 older Firefighters (FF) and 12 older Non-Firefighters (Non-FF), and a group of 6 young FF and 6 young Non-FF, following intermittent exercise in the heat. The participants, matched for age, body surface area, body composition, and VO(2peak), cycled 4 × 15 min at moderate-to-high heat production (400 W), each followed by 15-min rest, in dry [10-20% relative humidity (RH)] and humid (60% RH) heat (35 °C). Rectal temperature (T(re)) and heart rate (HR) were measured continuously, and blood samples at baseline (PRE) and following 60-min recovery (POST) were analyzed for Interleukin (IL)-6, Tumor Necrosis Factor (TNF-α), C-reactive protein (CRP), platelet count (PLT), and mean platelet volume (MPV). No differences were observed for T(re), HR, TNF-α, CRP, or PLT between the FF and Non-FF in either condition. The Non-FF had greater changes in IL-6 (Warm/Dry = +1.10 ± 0.18, Warm/Humid = +2.94 ± 0.74 pg mL(-1)), compared to the FF (Warm/Dry = +0.67 ± 0.17, Warm/Humid = +0.70 ± 0.33 pg mL(-1)), and MPV at PRE/POST compared to the FF. Between the young and older FF and Non-FF, no differences in T(re), HR, PLT, or MPV were observed, however, elevated CRP (Warm/Dry) in the older FF, and IL-6 (Warm/Dry) and TNF-α (both conditions) in the older Non-FF, were observed. The elevated IL-6 in the Non-FF is potentially indicative of increased strain in the Non-FF and/or adaptive changes in the FF due to the occupational demands.

How Might We Cure HIV?

Antiretroviral therapy (ART) does not eliminate HIV-1 from latently infected reservoirs, and this remains the critical obstacle to the eradication of infection. Although ART is effective in suppressing viral load, life-long ART is burdensome in many respects. Given expanding numbers of HIV-infected individuals on ART worldwide, there is an urgent need to examine the possibility that innovative therapies might eradicate infection, and obviate the need for life-long medical therapy for HIV-positive people around the world. Several approaches to eradicating the latent HIV reservoir and curing infection have been proposed and are under study. An initial strategy seeks to induce the expression of the latent integrated proviral genomes within resting CD4+ T cells, so that viral proteins or particles may be revealed and allow these cellular reservoirs to be cleared. The inducing agents that have been studied recently are inhibitors of histone deacetylase (HDAC) such as suberoylanilide hydroxamic acid (SAHA). Such induction of viral expression seems unlikely in itself to efficiently clear all latently infected cells. Therefore, it seems likely that parallel efforts to augment the HIV-specific immune response with specific immunotherapies or vaccination may be required. Recently, efforts to achieve immune augmentation by ex vivo expansion of viral specific cytotoxic T-cell lymphocytes derived from HIV-infected patients have yielded an augmented HIV-specific immune response in vivo, as have cellular vaccinations delivered by administration of dendritic cells. As HIV latency and the persistence of infection despite effective ART is multifactorial, the eradication of HIV infection may require multiple approaches.

Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures

Influenza viruses are global pathogens that infect approximately 10–20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent clinical phenotype seen in human LAIV inoculation.

Management of facial erythema of rosacea: What is the role of topical α-adrenergic receptor agonist therapy?

Several more recent advances have led to a better understanding of the pathophysiologic mechanisms involved in rosacea and therapeutic modalities used for treatment. Although the clinical features may vary among patients, there are some unifying mechanisms that appear to relate to the more common presentations of rosacea. Both neurovascular dysregulation and augmented immune detection and response appear to play central roles that lead to many of the signs and symptoms of rosacea. Diffuse central facial erythema is a very common finding that intensifies during flares and persists to varying degrees between flares. This background of facial redness occurs secondary to vasodilation and fixed vascular changes that develop over time. Physical modalities are commonly used to treat the erythema that persists as a result of fixed changes in superficial cutaneous vasculature that do not remit after treatment with agents whose mechanisms are active primarily against some of the inflammatory processes operative in rosacea (ie metronidazole, azelaic acid, tetracyclines). As enlarged superficial cutaneous vessels that contribute to the fixed background facial redness of rosacea remain vasoactive to sympathetic nervous system innervation, topical α-adrenergic receptor agonists, namely brimonidine and oxymetazoline, are currently under evaluation for the treatment of facial erythema of rosacea. This article focuses on the clinical differentiation of facial erythema of rosacea and its management.

The Transcription Factor T-bet Regulates Parasitemia and Promotes Pathogenesis duringPlasmodium bergheiANKA Murine Malaria

The pathogenesis of experimental cerebral malaria (ECM) is an immunologic process, mediated in part by Th1 CD4(+) T cells. However, the role of the Th1 CD4(+) T cell differentiation program on the ability to control parasitemia and susceptibility to ECM disease during blood stage malaria has never been assessed directly. Using the Plasmodium berghei ANKA murine model of ECM and mice deficient for the transcription factor T-bet (the master regulator of Th1 cells) on the susceptible C57BL/6 background, we demonstrate that although T-bet plays a role in the regulation of parasite burden, it also promotes the pathogenesis of ECM. T-bet-deficient (Tbx21(-/-)) mice had higher parasitemia than wild type controls did during the ECM phase of disease (17.7 ± 3.1% versus 10.9 ± 1.5%). In addition, although 100% (10/10) of wild type mice developed ECM by day 9 after infection, only 30% (3/10) of Tbx21(-/-) mice succumbed to disease during the cerebral phase of infection. Resistance to ECM in Tbx21(-/-) mice was associated with diminished numbers of IFN-γ-producing CD4(+) T cells in the spleen and a lower accumulation of CD4(+) and CD8(+) T cells in the brain. An augmented Th2 immune response characterized by enhanced production of activated GATA-3(+) CD4(+) T cells and elevated levels of the eotaxin, MCP-1, and G-CSF cytokines was observed in the absence of T-bet. Our results suggest that in virulent malarias, immune modulation or therapy resulting in an early shift toward a Th2 response may help to ameliorate the most severe consequences of malaria immunopathogenesis and the prospect of host survival.

NFκB induces overexpression of bovine FcRn

Among the many functions of the neonatal Fc receptor (FcRn) for IgG, it binds to IgG-opsonized antigen complexes and propagates their traffic into lysosomes where antigen processing occurs. We previously reported that transgenic (Tg) mice and rabbits that carry multiple copies and overexpress FcRn have augmented humoral immune responses. Nuclear factor-kappa B (NFκB) is a critical molecule in the signaling cascade in the immune response. NFκB induces human FcRn expression and our previous in silico analysis suggested NFκB binding sites in the promoter region of the bovine (b) FcRn α-chain gene (FCGRT). Here, we report the identification of three NFκB transcription binding sites in the promoter region of this gene using luciferase reporter gene technology, electromobility shift assay and supershift analysis. Stimulation of primary bovine endothelial cells with the Toll-like receptor-4 ligand lipopolysaccharide (LPS), which mediates its effect via NFκB, resulted in rapid upregulation of the bFcRn expression and a control gene, bovine E-selectin. This rapid bFcRn gene induction was also observed in the spleen of bFcRn Tg mice treated with intraperitoneally injected LPS, analyzed by northern blot analysis. Finally, NFκB-mediated bFcRn upregulation was confirmed at the protein level in macrophages isolated from the bFcRn Tg mice using flow cytometry with a newly developed FcRn specific monoclonal antibody that does not cross-react with the mouse FcRn. We conclude that NFκB regulates bFcRn expression and thus optimizes its functions, e.g., in the professional antigen presenting cells, and contributes to the much augmented humoral immune response in the bFcRn Tg mice.

Transgenic Rabbits That Overexpress the Neonatal Fc Receptor (FcRn) Generate Higher Quantities and Improved Qualities of Anti-Thymocyte Globulin (ATG)

Immune suppression with rabbit anti-thymocyte globulin (rATG) is a well-established therapeutic concept for preventing host rejection of transplanted organs and graft versus host disease. Increasing the efficiency of rATG production by reducing the number of animals would be highly beneficial to lower cost and to improve quality standards. We have developed transgenic (Tg) mice and rabbits that overexpress the neonatal Fc receptor (FcRn) and have shown an augmented humoral immune response in these animals. To test whether our FcRn Tg rabbits produced rATG more efficiently, we immunized them and their New Zealand White controls with live Jurkat cells. By day 21 after immunization, Tg animals produced significantly, 1.5 times higher amount of total IgG compared to their wt littermates. Also, the binding efficiency of Tg sera to Jurkat cells and their complement-mediated cytotoxicity was significantly higher. The purified Tg IgG preparation contained 2.6 the amount of Jurkat specific IgG as the wt preparation analyzed by complement-mediated lysis, suggesting greater antigen-specific B cell activation in the Tg rabbits. To test this hypothesis, immunization with ovalbumin and human α1-antitrypsin was performed, resulting in significantly greater numbers of antigen-specific B-cells in the FcRn Tg rabbits as compared with wt controls. The shift towards significantly larger populations of antigen-specific B cells relative to the non-specific B cell pool is further corroborated by our previous findings in FcRn Tg mice. Consequently, our FcRn Tg rabbits have the potential to offer substantial qualitative and quantitative improvements for the production of rATG and other polyclonal or monoclonal antibodies.

Augmented primary humoral immune response and decreased cell-mediated immunity by Murraya koenigii in rats

Murraya koenigii (Rutaceae) (curry patta: Hindi) of the family Rutaceae is used in the traditional Indian system of medicine for its immunomodulatory properties. The essential oil of the leaves of M. koenigii possesses antimicrobial, antifungal, and pesticidal activities and is used for the treatment of amebiasis, diabetes, and hepatitis. The present study was performed to evaluate the effect of M. koenigii on humoral and cell-mediated immune responses in rats. Aqueous extract of M. koenigii leaves was administered orally in a dose of 350 mg/kg. Cell-mediated immunity was assessed by measuring foot pad thickness following sensitization by injection of keyhole limpet hemocyanin and subsequent challenge by the same. Humoral immunity was assessed by measurement of hemagglutination titer to sheep red blood cells (SRBCs). In the humoral immune response, the administration of M. koenigii [350 mg/kg per os (p.o.)] from day 1 to day 7 after sensitization with SRBC on day 0 caused a significant increase in the primary anti-SRBC titer. However, the secondary immune response was decreased significantly (p<0.05) as shown by a decrease in secondary anti-SRBC titer measured on day 11 following a booster dose of antigen on day 8. In the delayed-type hypersensitivity test, M. koenigii (350 mg/kg, p.o.), when administered for 14 days, produced a significant (p<0.05) decrease in foot pad thickness when compared with the control group. Thus, these results suggest that oral administration of M. koenigii augments primary humoral immune response and decreases cell-mediated immunity.

Investigating genes and micro-RNAs that may predict clinical benefits of anti-CTLA-4 therapy.

3043 Background: Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, ipilimumab, has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of ipilimumab for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4 therapy. In order to identify genes, microRNAs, and signaling pathways that are modulated by anti-CTLA-4, which may be used for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4+T cells from patients treated with anti-CTLA-4 for changes in gene and microRNA expression profiles. Methods: On an IRB-approved phase Ia presurgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of ipilimumab at 10 mg/kg at weeks 1 and 4. Pre-therapy and post-therapy blood samples were collected for CD4+ T cell enrichment by using the T cell isolation kit from Miltenyi Biotec (Auburn, CA). RNA and microRNA were isolated from purified CD4+T cells using Qiagen RNA isolation kits for Affymetrix microarray and micoRNA array analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. Results: Anti-CTLA-4 treatment resulted in modulation of differentially expressed genes (DEGs). After two doses of treatment, anti-CTLA-4 significantly changed expression of a total of 289 DEGs. Further pathway analyses indicated that anti-CTLA-4 induced a variety of pathways involved in cell proliferation and immune modulation, including PI3K/AKT, MAP/ERK, and IFN/JAK-STAT pathways. We have also identified 9 microRNAs that potentially regulate the expression of genes changed by anti-CTLA-4 therapy. Conclusions: Anti-CTLA-4 treatment results in modulation of multiple genes, microRNAs, and pathways, which likely play important roles in anti-tumor immune responses. We are currently testing a number of these identified genes and microRNAs as potential predictive biomarkers for anti-CTLA-4 therapy in a small cohort of patients who had complete response vs. progression of disease after anti-CTLA-4 therapy.

A randomized phase II study evaluating the optimal sequencing of sipuleucel-T and androgen deprivation therapy (ADT) in biochemically recurrent prostate cancer (BRPC): Immune results.

5016 Background: ADT is a standard treatment for men with BRPC after failure of local therapy, and has immunomodulatory effects. Sipuleucel-T is an autologous cellular immunotherapy approved for asymptomatic/minimally symptomatic metastatic castrate resistant prostate cancer. The STAND trial (NCT01431391) aimed to evaluate optimal sequencing of sipuleucel-T and ADT in men with BRPC at high risk for metastases (ie PSA doubling time ≤12 mo). Methods: Men were randomized (1:1) to Arm 1: sipuleucel-T followed by ADT (2 wks after 3rd infusion); or Arm 2: ADT (3 mo lead in) followed by sipuleucel-T. All men had 3 doses of sipuleucel-T and 12 mo of ADT (45 mg leuprolide SQ at 6 mo intervals). The primary endpoint is cellular immune response (ELISPOT to PA2024 [PAP-GMCSF]). Secondary endpoints are humoral and cytokine responses, product parameters and safety. Results: 68 men were randomized. Preliminary data show higher levels of serum cytokines in Arm 2 vs Arm 1, with a pattern suggesting a mixed TH1/TH2 cellular immune response; elevations were seen in TH1 (IFNγ, IL 12), TH2 (IL 4, 5, 10, 13) and TH17 (IL 17) subsets (all P&lt;.05). The increase in TH1 cytokines was consistent with a trend toward higher PA2024-specific ELISPOT responses 2 wk after the 3rd sipuleucel-T infusion in Arm 2 vs Arm 1 (40.5 vs 12.8 spots; P=.086), suggesting increased T cell activation in Arm 2. Antigen-specific humoral responses were induced in both arms with no differences yet observed between arms. Sipuleucel-T product parameters were roughly equivalent in both arms with APC activation data indicating a robust prime-boost effect. Conclusions: While confirmation is required, these preliminary data suggest that tumor-specific T cell responses and broad based immune responses are augmented when sipuleucel-T is given after rather than before ADT initiation. These data are consistent with preclinical studies showing that ADT enhances T cell activity, and provide preliminary evidence that combining ADT with sipuleucel-T may augment adaptive immunity. Further follow up will determine whether augmented immune responses correlate with clinical parameters (eg PSA recurrence). Clinical trial information: NCT01431391.

FcRn overexpression in transgenic animals results in robust immune response (P1069)

Abstract Our previous studies have shown that overexpression of the neonatal Fc receptor (FcRn) in transgenic animals (mice and rabbits) leads to (1) improved IgG protection; (2) larger numbers of antigen specific B cells; (3) generation of antibodies against weakly immunogenic antigens; (4) generation of antibodies against highly conserved antigens (breaking immune tolerance); (5) increased humoral diversity and (6) higher levels of circulating antibodies. FcRn transgenesis thus confers a number of practical benefits, including faster antibody production, higher antibody yields and improved generation of hybridomas for monoclonal antibody production. To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized our FcRn transgenic animals. Our analyses showed strong expression of the FcRn transgene in professional antigen presenting cells and we also found that overexpression of the FcRn significantly improves antigen presentation in case of antigen-IgG immune complexes. We have recently analyzed several key factors in the maturation of the immune response, including the structure of the spleen, number and structure of germinal centers, the level of T cell activation and the affinity of the generated antibodies. These results provide further insights of those mechanisms that are involved in the augmented humoral immune response in transgenic animals that overexpress FcRn.

A study of genes and microRNAs that may predict clinical responses to anti-CTLA-4 therapy.

285 Background: Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, Ipilimumab (BMS), has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of Ipilimumab for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4 therapy. In order to identify genes, microRNAs, and signaling pathways that are modulated by anti-CTLA-4, which may be used for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4+ T cells from patients treated with anti-CTLA-4 for changes in gene and microRNA expression profiles. Methods: On an IRB-approved Phase Ia presurgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of Ipilimumab at 10 mg/kg at weeks 1 and 4. Pre-therapy and post-therapy blood samples were collected for CD4+ T cell enrichment by using the T cell isolation kit from Miltenyi Biotec (Auburn, CA). RNA and microRNA were isolated from purified CD4+ T cells using Qiagen RNA isolation kits for Affymetrix microarray and micoRNA array analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. Results: Ipilimumab treatment resulted in modulation of differentially expressed genes (DEGs). After two doses of treatment, Ipilimumab significantly changed expression of a total of 289 DEGs. Further pathway analyses indicated that Ipilimumab induced a variety of pathways involved in cell proliferation and immune modulation, including PI3K/AKT, MAP/ERK, and IFN/JAK-STAT pathways. We have also identified 9 microRNAs that potentially regulate the expression of genes changed by anti-CTLA-4 therapy. Conclusions: Ipilimumab treatment results in modulation of multiple genes, microRNAs, and pathways, which likely play important roles in anti-tumor immune responses. We are currently testing a number of these identified genes and microRNAs as potential predictive biomarkers for anti-CTLA-4 therapy in a small cohort of patients who had complete response vs. progression of disease after anti-CTLA-4 therapy.

NKT Cells as an Ideal Anti-Tumor Immunotherapeutic

Human natural killer T (NKT) cells are characterized by their expression of an invariant T cell antigen receptor α chain variable region encoded by a Vα24Jα18 rearrangement. These NKT cells recognize α-galactosylceramide (α-GalCer) in conjunction with the MHC class I-like CD1d molecule and bridge the innate and acquired immune systems to mediate efficient and augmented immune responses. A prime example of one such function is adjuvant activity: NKT cells augment anti-tumor responses because they can rapidly produce large amounts of IFN-γ, which acts on NK cells to eliminate MHC negative tumors and also on CD8 cytotoxic T cells to kill MHC positive tumors. Thus, upon administration of α-GalCer-pulsed DCs, both MHC negative and positive tumor cells can be effectively eliminated, resulting in complete tumor eradication without tumor recurrence. Clinical trials have been completed in a cohort of 17 patients with advanced non-small cell lung cancers and 10 cases of head and neck tumors. Sixty percent of advanced lung cancer patients with high IFN-γ production had significantly prolonged median survival times of 29.3 months with only the primary treatment. In the case of head and neck tumors, 10 patients who completed the trial all had stable disease or partial responses 5 weeks after the combination therapy of α-GalCer-DCs and activated NKT cells. We now focus on two potential powerful treatment options for the future. One is to establish artificial adjuvant vector cells containing tumor mRNA and α-GalCer/CD1d. This stimulates host NKT cells followed by DC maturation and NK cell activation but also induces tumor-specific long-term memory CD8 killer T cell responses, suppressing tumor metastasis even 1 year after the initial single injection. The other approach is to establish induced pluripotent stem (iPS) cells that can generate unlimited numbers of NKT cells with adjuvant activity. Such iPS-derived NKT cells produce IFN-γ in vitro and in vivo upon stimulation with α-GalCer/DCs, and mediated adjuvant effects, suppressing tumor growth in vivo.