Search

Thyroid cancer recurrence following curative thyroidectomy is associated with increased morbidity and mortality, but current surveillance strategies are inadequate for early detection. Prior studies indicate that tissue glycosylation is altered in thyroid cancer, but the utility of serum glycosylation in thyroid cancer surveillance remains unexplored. We therefore assessed the potential utility of altered serum glycomic profile as a tumor-specific target for disease surveillance in recurrent thyroid cancer.We employed banked serum samples from patients with recurrent thyroid cancer post thyroidectomy and healthy controls. N-glycans were enzymatically released from serum glycoproteins, labeled via permethylation, and analyzed by MALDI-TOF mass spectrometry. Global level and specific subtypes of glycan structures were compared between patients and controls.We evaluated 28 independent samples from 13 patients with cancer recurrence and 15 healthy controls. Global features of glycosylation, including N-glycan class and terminal glycan modifications were similar between groups, but three of 35 individual glycans showed significant differences. The three glycans were biosynthetically related biantennary core fucosylated N-glycans that only varied by the degree of galactosylation (G0F, G1F, and G2F; G: galactose, F: fucose). The ratio of G0F:G1F that captures reduced galactosylation was observed in patients samples but not in healthy controls (p = 0.004) and predicted thyroid cancer recurrence (AUC = 0.82, CI 95% = 0.64-0.99).Altered N-glycomic profile was associated with thyroid cancer recurrence. This serum-based biomarker would be useful as an effective surveillance tool to improve the care and prognosis of thyroid cancer after prospective validation.

Alzheimer’s disease (AD) is a neurodegenerative disease with only late-stage detection; thus, diagnosis is made when it is no longer possible to treat the disease, only its symptoms. Consequently, this often leads to caregivers who are the patient’s relatives, which adversely impacts the workforce along with severely diminishing the quality of life for all involved. It is, therefore, highly desirable to develop a fast, effective and reliable sensor to enable early-stage detection in an attempt to reverse disease progression. This research validates the detection of amyloid-beta 42 (Aβ42) using a Silicon Carbide (SiC) electrode, a fact that is unprecedented in the literature. Aβ42 is considered a reliable biomarker for AD detection, as reported in previous studies. To validate the detection with a SiC-based electrochemical sensor, a gold (Au) electrode-based electrochemical sensor was used as a control. The same cleaning, functionalization and Aβ1–28 antibody immobilization steps were used on both electrodes. Sensor validation was carried out by means of Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) aiming to detect an 0.5 µg·mL−1 Aβ42 concentration in 0.1 M buffer solution as a proof of concept. A repeatable peak directly related to the presence of Aβ42 was observed, indicating that a fast SiC-based electrochemical sensor was constructed and may prove to be a useful approach for the early detection of AD.

Background Purpose: The combination of a mammalian target of rapamycin inhibitor and lenalidomide showed enhanced preclinical cytotoxicity. We conducted a phase 1 study in advanced solid tumour patients to assess safety, efficacy and pharmacodynamic (PD) outcomes. Methods We employed a 3+3 dose escalation design to establish the safety and recommended phase 2 doses (RP2D) of daily everolimus and lenalidomide in patients with advanced solid tumours. The starting doses were 5 and 10 mg, respectively, with planned escalation to maximum single-agent doses of 10 and 25 mg in the absence of dose-limiting toxicity. PD endpoints of lymphocyte subsets and immune cytokines were assessed in peripheral blood using multiparameter flow cytometry and LUMINEX assay. Efficacy was evaluated by cross-sectional imaging after every two cycles of treatment. Results The study enrolled 44 patients, median age of 58 years and 28 males (63.6%). The RP2D was established as 10 and 25 mg daily continuously for everolimus and lenalidomide. Common (>5%) grade ≥3 adverse events included rash (19%), neutropenia (19%), hypokalaemia (11%) and fatigue (9%). Best efficacy outcomes in 36 evaluable patients were partial response in 5 (13.8%), stable disease in 24 (55.8%) and progressive disease in 7 (19.4%) patients. PD assessment revealed significant association of cytokine levels (interleukin-2 (IL2), IL21 and IL17), baseline activated and total CD8+ lymphocytes and change in B cell lymphocytes and activated NK cells with clinical benefit. Conclusions The study demonstrated the safety of everolimus and lenalidomide with promising efficacy signal in thyroid and adenoid cystic cancers. Clinical Trial Registration NCT01218555

Parasite resistance to antimalarial drugs poses a serious threat to malaria control. The WorldWide Antimalarial Resistance Network (WWARN) aims to provide a collaborative platform to support the global malaria research effort. Here, we describe the “WWARN clinical trials publication library,” an open-access, up-to-date resource to streamline the synthesis of antimalarial safety and efficacy data. A series of iteratively refined database searches were conducted to identify prospective clinical trials assessing antimalarial drug efficacy with at least 28 days of follow-up. Of approximately 45,000 articles screened, 1,221 trials published between 1946 and 2018 were identified, representing 2,339 treatment arms and 323,819 patients. In trials from endemic locations, 75.7% (787/1,040) recruited patients with Plasmodium falciparum, 17.0% (177/1,040) Plasmodium vivax, 6.9% (72/1,040) both, and 0.4% (4/1,040) other Plasmodium species; 57.2% (585/1,022) of trials included under-fives and 5.3% (55/1,036) included pregnant women. In Africa, there has been a marked increase in both P. falciparum and P. vivax studies over the last two decades. The WHO-recommended artemisinin-based combination therapies alone or with a gametocidal drug were assessed in 39.5% (705/1,783) of P. falciparum treatment arms and 10.5% (45/429) of P. vivax arms, increasing to 78.0% (266/341) and 22.9% (27/118), respectively, in the last five years. The library is a comprehensive, open-access tool that can be used by the malaria community to explore the collective knowledge on antimalarial efficacy (available at https://www.wwarn.org/tools-resources/literature-reviews/wwarn-clinical-trials-publication-library). It is the first of its kind in the field of global infectious diseases, and lessons learnt in its creation can be adapted to other infectious diseases.

BackgroundImmune checkpoint blockade (ICB) targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein 4 has achieved modest clinical activity as salvage therapy in relapsed small cell lung cancer (SCLC). We conducted this signal-finding study to assess the efficacy of ICB with or without radiation in relapsed SCLC.MethodsPatients with relapsed SCLC and ≤2 previous lines of therapy were randomized to (1) arm A: durvalumab (D) 1500 mg/tremelimumab (T) 75 mg (intravenously every 4 weeks without stereotactic body radiation therapy (SBRT)) or (2) arm B: immune-sensitizing SBRT to one selected tumor site (9 Gy × 3 fractions) followed by D/T. Treatment continued until progression or a maximum of 12 months. The co-primary endpoints of the study were overall response rate (ORR) and progression-free survival (PFS). We evaluated circulating lymphocyte repertoire in serial peripheral blood samples and tumor infiltrating lymphocytes (TILs) from on-treatment biopsies as pharmacodynamic markers.ResultsEighteen patients were randomized to arms A and B (n=9 each): median age 70 years; 41.2% women. The median PFS and ORR were 2.1 months and 0% in arm A and 3.3 months and 28.6% in arm B. The median overall survival (OS) was 2.8 months in arm A and 5.7 months in arm B (p=0.3772). Pooled efficacy of D/T±SBRT in 15 Response evaluation criteria in solid tumors (RECIST) evaluable patients across both arms showed the best ORR in terms of partial response in 13.3%, stable disease in 26.6% and progressive disease in 60.0%; the overall median PFS and OS were 2.76 and 3.9 months. The most common adverse events were grade 1 fatigue (66%) and grade 1 elevated amylase (56%) in arm A, and grade 1 fatigue (56%) and pain (44%) in arm B. There was a significant increase in activated CD8(+)ICOS+ T cells (p=0.048) and a reduction in naïve T cells (p=0.0454) in peripheral blood following treatment, along with a significant amount of activated CD8+ICOS+ T cells in TILs from responders.ConclusionsThe D/T combination with and without SBRT was safe but did not show sufficient efficacy signal in relapsed SCLC. Changes in peripheral blood lymphocyte and TILs were consistent with an immunologic response.Trial registration numberNCT02701400.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2−). Previous chemical rescue studies identified a putative FeIII-O2•− intermediate that precedes substrate oxidation in Mus musculus cysteine dioxygenase (Mm CDO). Given that a similar reactive intermediate has been identified in the extradiol dioxygenase 2, 3-HCPD, it is conceivable that these enzymes share other mechanistic features with regard to substrate oxidation. To explore this possibility, enzymatic reactions with Mm CDO (as well as the bacterial 3-mercaptopropionic acid dioxygenase, Av MDO) were performed using a substrate analogue (2-mercaptoaniline, 2ma). This aromatic thiol closely approximates the catecholic substrate of homoprotocatechuate of 2, 3-HPCD while maintaining the 2-carbon thiol-amine separation preferred by Mm CDO. Remarkably, both enzymes exhibit 2ma-gated O2-consumption; however, none of the expected products for thiol dioxygenase or intra/extradiol dioxygenase reactions were observed. Instead, benzothiazoles are produced by the condensation of 2ma with aldehydes formed by an off-pathway oxidation of primary alcohols added to aqueous reactions to solubilize the substrate. The observed oxidation of 1°-alcohols in 2ma-reactions is consistent with the formation of a high-valent intermediate similar to what has been reported for cytochrome P450 and mononuclear iron model complexes.

The expression of checkpoint blockade molecules PD-1, PD-L1, CTLA-4, and foxp3+CD25+CD4+ T cells (Tregs) regulate donor T cell activation and graft-vs-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT). Detailed kinetics of PD-1-, CTLA-4-, and PD-L1 expression on donor and host cells in GvHD target organs have not been well studied. Using an established GvHD model of allo-HSCT (B6 → CB6F1), we noted transient increases of PD-1- and CTLA-4-expressing donor CD4+ and CD8+ T cells on day 10 post transplant in spleens of allo-HSCT recipients compared with syngeneic HSCT (syn-HSCT) recipients. In contrast, expression of PD-1- and CTLA-4 on donor T cells was persistently increased in bone marrow (BM) of allo-HSCT recipients compared with syn-HSCT recipients. Similar differential patterns of donor T cell immune response were observed in a minor histocompatibility (miHA) mismatched transplant model of GvHD. Despite higher PD-1 and CTLA-4 expression in BM, numbers of foxp3+ T cells and Tregs were much lower in allo-HSCT recipients compared with syn-HSCT recipients. PD-L1-expressing host cells were markedly decreased concomitant with elimination of residual host hematopoietic elements in spleens of allo-HSCT recipients. Allo-HSCT recipients lacking PD-L1 rapidly developed increased serum inflammatory cytokines and lethal acute GvHD compared with wild-type (WT) B6 allo-HSCT recipients. These data suggest that increased expression of checkpoint blockade molecules PD-1 and CTLA-4 on donor T cells is not sufficient to prevent GvHD, and that cooperation between checkpoint blockade signaling by host cells and donor Tregs is necessary to limit GvHD in allo-HSCT recipients.

Cytomegalovirus (CMV) infection following allogeneic bone marrow transplant (allo-BMT) is controlled by donor-derived cellular immunity. Vasoactive intestinal peptide (VIP) suppresses Th1 immunity. We hypothesized that blocking VIP-signaling would enhance anti-CMV immunity in murine recipients of allo-BMT. Recipients were transplanted with bone marrow (BM) and T-cells from major histocompatibility complex (MHC)-mismatched VIP-knockout (KO) or wild-type donors, and treated with 7 daily subcutaneous injections of VIPhyb (peptidic VIP-antagonist) or phosphate-buffered saline (PBS). Genetic and pharmacological blockade of VIP-signaling protected allo-BMT recipients from lethal murine CMV (mCMV) infection, improving survival without increasing graft-versus-host disease. Mice treated with VIPhyb or transplanted with VIP-KO allografts had significantly lower viral loads, increased numbers of mCMV-M45-peptide-MHC-tetramer(+) CD8(+) T-cells, with lower PD-1 expression, and enhanced primary and secondary cellular immune responses after mCMV infection than did PBS-treated mice. These results demonstrate that administration of a VIP antagonist after allo-BMT is a promising safely therapeutic approach to enhance antiviral cellular immunity.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2b → CB6F1 and H-2K → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4+CD25+Foxp3+ regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.

Vasoactive intestinal peptide (VIP) induces regulatory dendritic cells (DC) in vitro that inhibit cellular immune responses. We tested the role of physiological levels of VIP on immune responses to murine CMV (mCMV) using VIP-knockout (VIP-KO) mice and radiation chimeras engrafted with syngenic VIP-KO hematopoietic cells. VIP-KO mice had less weight loss and better survival following mCMV infection compared with wild-type (WT) littermates. mCMV-infected VIP-KO mice had lower viral loads, faster clearance of virus, with increased numbers of IFN-γ+ NK and NKT cells, and enhanced cytolytic activity of NK cells. Adaptive antiviral cellular immunity was increased in mCMV-infected VIP-KO mice compared with WT mice, with more Th1/Tc1-polarized T cells, fewer IL-10+ T cells, and more mCMV-M45 epitope peptide MHC class I tetramer+ CD8+ T cells (tetramer+ CD8 T cells). mCMV-immune VIP-KO mice had enhanced ability to clear mCMV peptide-pulsed target cells in vivo. Enhanced antiviral immunity was also seen in WT transplant recipients engrafted with VIP-KO hematopoietic cells, indicating that VIP synthesized by neuronal cells did not suppress immune responses. Following mCMV infection there was a marked upregulation of MHC-II and CD80 costimulatory molecule expression on DC from VIP-KO mice compared with DC from WT mice, whereas programmed death-1 and programmed death ligand-1 expression were upregulated in activated CD8+ T cells and DC, respectively, in WT mice, but not in VIP-KO mice. Because the absence of VIP in immune cells increased innate and adaptive antiviral immunity by altering costimulatory and coinhibitory pathways, selective targeting of VIP signaling represents an attractive therapeutic target to enhance antiviral immunity.

When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform theta (PKCtheta), a key regulator of TCR signaling. In contrast, PKCtheta was required for alloreactivity and GVHD induction. Furthermore, absence of PKCtheta raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCtheta-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCtheta is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.

Inflammatory bowel disease (IBD) severity is positively correlated with cytomegalovirus (CMV) infection. This may reflect CMV triggering and/or exacerbating flares of IBD and/or IBD or immunosuppressive drugs administered to patients with IBD increasing susceptibility to CMV infection. Herein, we performed studies in mice to investigate these possibilities. Mice administered murine CMV (MCMV) developed signs of acute viral infection (malaise and weight loss) and had MCMV loads that were readily detected in numerous organs including the intestine. By 4 weeks, these mice manifested a "latent" infection in which MCMV levels were low but detectable by PCR. Such MCMV infection did not induce acute colitis in either wild-type mice or IL-10(-/-) mice, which are highly prone to developing colitis. However, underlying MCMV infection in an acute or latent state exacerbated the severity of colitis induced by dextran sodium sulfate (DSS). Such potentiation of DSS colitis by latent MCMV infection seemed to occur without viral reactivation. Whereas initial MCMV infection induced acute alterations in serum and intestinal cytokines, such cytokine levels returned to baseline before administration of DSS. However, the initial infection resulted in lasting elevation of antibodies to commensal bacterial antigens, suggesting that MCMV infection may have potentiated colitis via priming of the intestinal immune response to gut microbiota. Thus, underlying CMV infection can alter mucosal immunity, potentially increasing the tendency of CMV-infected hosts to develop colitis.

We have previously shown that amotosalen-treated splenocytes rescued allorecipients from a lethal dose of mouse CMV (MCMV) administered on day 0 in experimental parent C57BL/6→CB6F1 allogeneic bone marrow transplant. In this study, we investigated the mechanism of antiviral activity of amotosalen-treated donor splenocytes when sublethal MCMV infections were administered 7 days posttransplant. Recipients of 3 × 106 untreated splenocytes were used as control. Following MCMV infection, recipients of untreated splenocytes had 40% early mortality due to acute graft-vs-host disease compared with no deaths among recipients of 10 × 106 treated splenocytes. However, recipients of both types of donor splenocytes effectively cleared MCMV from their liver. Like the untreated CD8+ T cells, amotosalen-treated CD8+ T cells equally retained their in vivo CTL activity against MCMV early peptide-pulsed targets and expressed similar levels of granzyme B within 11 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with both donor spleen- and host-derived anti-MCMV CD8+ T cells in their blood and lymphoid organs, with significantly higher numbers of host-derived CD4−CD8− (double negative) T cells in the spleens of recipients of treated splenocytes compared with the recipients of untreated splenocytes. Additionally, recipients of amotosalen-treated splenocytes had lower levels of serum IFN-γ and TNF-α in response to MCMV infection compared with untreated recipients. Thus, adoptive immunotherapy with treated T cells is a novel therapeutic approach that facilitates hematopoietic engraftment and permits antiviral immunity of both donor and host T cells without graft-vs-host disease.

Chronic graft-versus-host disease (cGvHD) is associated with functional immunodeficiency and an increased risk of opportunistic infections in allogeneic bone marrow transplantation (BMT). We used a parent to F1 model of allogeneic BMT to test the hypothesis that cGvHD leads to impaired antigen-specific antiviral immunity and compared BM transplant recipients with cGvHD to control groups of allogeneic BM transplant recipients without GvHD. Mice with and without cGvHD received a nonlethal dose of murine cytomegalovirus (MCMV) +100 days after transplantation. Recipients with cGvHD had more weight loss and higher viral loads in the spleen and liver. MCMV infection led to greater than 25-fold expansion of donor spleen–derived MCMV peptide–specific tetramer-positive CD8+ T cells in blood of transplant recipients with and without cGvHD, but mice with cGvHD had far fewer antigen-specific T cells in peripheral tissues and secondary lymphoid organs. The immunosuppression associated with cGvHD was confirmed by vaccinating transplant recipients with and without cGvHD using a recombinant Listeria expressing MCMV early protein (Lm-MCMV). Secondary adoptive transfer of lymphocytes from donor mice with or without cGvHD into lymphopenic congenic recipients showed that cGvHD impaired tissue-specific homing of antigen-specific T cells. These results indicate that cGvHD causes an intrinsic immunosuppression and explain, in part, the functional immunodeficiency in allogeneic transplant recipients.

Delayed reconstitution of cellular immunity following T-cell-depleted, CD34-enriched, allogeneic hematopoietic progenitor cell transplantation (HPCT) is the major cause of morbidity and mortality following haploidentical transplantation in adults. This is illustrated in our recent study of 28 high-risk adult patients (median age 31) who were treated with conditioning regimens containing antithymocyte globulin (ATG) before T-cell-depleted, CD34-enriched allogeneic HPCT. Overall mortality was 93% (26/28 patients) with a median survival of 4 months posttransplant. Poor cellular immune reconstitution contributed to death of 21/28 patients, with eight deaths due to opportunistic infections and seven deaths due to relapse. While recovery of normal numbers of circulating NK cells and B-cells occurred within the first 1-2 months posttransplant, recovery of normal numbers of blood T-cells was suppressed for more than 1 year. The mean half-life of active ATG levels in serum was 6 days; rapid clearance suggested that residual ATG did not contribute to the delay of posttransplant T-cell reconstitution. Rapid T-cell reconstitution was seen only in younger patients, indicating that poor thymic function and the absence of T-cells in the graft are the major causes of delayed recovery of cellular immunity. Improved cellular immunity after T-cell-depleted haploidentical HPCT will thus require novel strategies to adoptively transfer antigen specific donor T-cells without inducing lethal graft-versus-host disease (GvHD). This problem has been addressed in a preclinical murine model of MHC-mismatched bone marrow transplantation. Donor T-cells treated ex vivo with fludarabine or a UVA light-activated psoralen compound (amotosalen) have a markedly reduced ability to induce GvHD, yet the treated T-cells confer protection against murine cytomegalovirus and an infused leukemic cell line. Polyclonal donor T-cells reconstituted the blood and lymphoid compartments posttransplant and expanded in vivo. Derivatives of ex-vivo-treated donor T-cells retained the ability to produce cytokines and proliferate in response to antigen challenge. The mechanism of reduced GvHD potential of ex-vivo-treated T-cells appears to be selection of a subset of memory donor T-cells that do not initially home to secondary lymphoid organs and have reduced capacity for producing inflammation in the immediate posttransplant period. Direct selection of the memory subset by high-speed FACS confirmed the improved therapeutic index in the murine model system. Preclinical data indicate the feasibility of treating human T-cells with fludarabine, psoralen, or direct selection based upon the memory phenotype to efficiently produce a population of polyclonal donor T-cells with reduced GvHD activity. A planned clinical phase 1 trial of adoptive therapy utilizing ex vivo psoralen-treated donor T-cells in recipients of T-cell-depleted haploidentical HPCT is presented.

A major goal of our research is to reduce the graft-vs-host disease (GVHD) activity of allogeneic donor T cells in bone marrow transplantation (BMT), while preserving graft-vs-tumor (GVT) effects. Using ex vivo immunosuppressive strategies and cell-separation techniques to modulate the graft prior to transplantation, we examined the roles of different donor immune cells on GVHD and GVT effects in allogeneic mouse models. Our results demonstrate that donor-memory CD4 T cells facilitate posttransplant immunological reconstitution without causing GVHD, whereas transplantation of equal numbers of donor naïve CD4 T cells leads to fatal GVHD. The initial events of donor T cells interacting with antigen-presenting cells (APCs) in the transplant recipient appear to be critical to the development of GVT, GVHD, or anergy to alloantigens. In the setting of clinical BMT, increased numbers of donor type 2 dendritic cells (DCs) were associated with an increased rate of posttransplant relapse, and decreased rates of chronic GVHD. In a mouse transplant model, manipulation of the DC content of bone marrow grafts was achieved by depletion of CD11b+ cells. Mice transplanted with CD11b- depleted marrow showed enhanced posttransplant expansion of memory T cells with markedly improved GVT activity and limited GVHD compared to recipients of unmanipulated marrow. A model that differentiates GVT from GVHD based on interaction of T-cell subsets with DC subsets is proposed.

Infusion of donor antiviral T cells can provide protective immunity for recipients of hemopoietic progenitor cell transplants, but may cause graft-vs-host disease (GVHD). Current methods of separating antiviral T cells from the alloreactive T cells that produce GVHD are neither routine nor rapid. In a model of lethal murine CMV (MCMV) infection following MHC-mismatched bone marrow transplantation, infusion of MCMV-immune donor lymphocytes pretreated with the DNA cross-linking compound amotosalen prevented MCMV lethality without producing GVHD. Although 95% of mice receiving 30 × 106 pretreated donor lymphocytes survived beyond day +100 without MCMV disease or GVHD, all mice receiving equivalent numbers of untreated lymphocytes rapidly died of GVHD. In vitro, amotosalen blocked T cell proliferation without suppressing MCMV peptide-induced IFN-γ production by MCMV-primed CD8+ T cells. In vivo, pretreated lymphocytes reduced hepatic MCMV load by 4-log10 and promoted full hemopoietic chimerism. Amotosalen-treated, MCMV tetramer-positive memory (CD44high) CD8+ T cells persisted to day +100 following infusion, and expressed IFN-γ when presented with viral peptide. Pretreated T cells were effective at preventing MCMV lethality over a wide range of concentrations. Thus, amotosalen treatment rapidly eliminates the GVHD activity of polyclonal T cells, while preserving long-term antiviral and graft facilitation effects, and may be clinically useful for routine adoptive immunotherapy.

Objective: The purpose of this paper is to assess the clinical effectiveness of percutaneous laser disc decompression (PLDD) for patients with radicular pain due to intervertebral disc hernia and to describe our experience in this field in Bangladesh. Background: PLDD is a procedure in which herniated intervertebral discs are treated by the reduction of intradiscal pressure by laser energy. This is introduced by a needle inserted into the nucleus pulposus under local anesthesia and fluoroscopic monitoring. Small volume nucleus vaporization results in a sharp fall in intradiscal pressure, with the consequent migration of herniation away from the nerve root. PLDD has been reported to be a safe, effective, minimally invasive treatment option for patients with a herniated disc. Materials and methods: This observational random prospective analysis was carried out during the period from November 2007 to December 2011. PLDD was performed on 4622 herniated discs of 2580 patients (1420, male; 1160, female), whose age ranged between 18 and 85 years. A total of 47 cases were failed back surgery syndrome (FBSS). All patients were followed up (ranging from 1 month to 36 months, with a median of 12 months) and were evaluated according to MacNab criteria. Results: Amongst the 2580 patients, 2296 (89.0 % ) reported immediate pain relief, 230 (8.9 % ) patients were uncertain and the remaining 54 (2.1 % ) did not have immediate pain relief. In the subsequent follow up, according to MacNab criteria a good response was seen in 1935 (75.0 % ) cases and a fair response in 335 (13.0 % ) cases. The response to the treatment was poor in 310 patients (12.0 % ). A second session of PLDD was performed on 292 (11.3 % ) patients made up of 162 patients (6.3 % ) due to multi-level ( > 2 discs) prolapse, 102 patients (3.9 % ) due to an extensive prolapse of one or two discs and 28 patients (1.0 % ) because of a relapse. In two cases of a prolapsed dorsal spine ( T 7 – 8 &T 8 – 9 ) a good response was obtained without any complications. All cases of FBSS showed a good and fair response. Conclusion: PLDD is an effective and minimally invasive procedure which has almost no side effects or complications, can be performed under local anesthesia, results in no scarring or spinal instability, and still does not hinder the scope of open surgery in failed cases. PLDD is also effective in cases of FBSS.

We have previously shown that highly purified cGMP grade recombinant flagellin, CBLB502, a TLR5 agonist, protected allo-HSCT recipients from GvHD without compromising anti-CMV immunity of donor T cells in vivo. A single prophylactic dose (25mg/mouse i.p) of CBLB502 in wild type C57BL/6 (B6) mice also enhanced anti-CMV immunity of NK cells against CMV. CMV is a potential opportunistic infectious agent and most often causes severe life-threatening clinical complications in immunocompromized patients, particularly in cancer patients treated with allogenic bone marrow transplantation (allo-BMT). A mechanism by which CBLB502 protected host against CMV infection has not been well investigated. Transcription factors ROR gammaT, T-bet and EOMES are known to control immune responses of innate and adaptive immunity in vivo. In this study, we have explored the transcription factors ROR gammaT, T-bet and EOMES expressing NK and T cells in CBLB502-treated and MCMV-infected B6 mice. Total numbers of spleen cells increased by 1.7-fold within 48 hours of CBLB502 treatment (day 0 MCMV infection) and by 2.3-fold on day 3 after MCMV infection compared with PBS-treated MCMV-infected mice. T-bet expressing CD8+ T cells (not CD4+ T cells) increased significantly on day 0 of mCMV infection, 48hrs after CBLB502 treatment (CD4, p=0.1; CD8, p90% of CMV-specific CD8+ T cells had persistent expression of T-bet even after 30 days post CMV infection. In contrast, CBLB502 treatment did not have a significant effect on the numbers of ROR gammaT, T-bet and EOMES expressing NK cells compared with PBS-treated mice. These data suggest that 1) CBLB502-induced anti-CMV immunity of T cells is persistently maintained through increased T-bet expression but not by EOMES expression; and 2) CBLB502-induced anti-viral immune regulation is not maintained through transcription factor ROR gammaT. 3) CBLB502-induced increased anti-CMV immunity of NK cells is independent of transcription factors ROR gammaT, T-bet and EOMES. Taken together, these data support the early peri-transplant use of TLR5 agonists as a novel method to enhance antigen-specific anti-viral immunity in recipients of allo-BMT. Disclosures No relevant conflicts of interest to declare.

Routine methods to maximize the graft-versus-leukemia (GvL) activity of allogeneic hematopoietic stem cell transplantation (HSCT) without the detrimental effects of graft-versus-host disease (GvHD) are lacking. Depletion or inhibition of alloreactive T cells is partially effective in preventing GvHD, but usually leads to decreased GvL activity. The current model for the pathophysiology of acute GvHD describes a series of immune pathways that lead to activation of donor T cells and inflammatory cytokines responsible for tissue damage in acute GvHD. This model does not account for how allotransplant can lead to GvL effects without GvHD, or how the initial activation of donor immune cells may lead to counter-regulatory effects that limit GvHD. In this review, we will summarize new findings that support a more complex model for the initiation of GvHD and GvL activities in allogeneic HSCT, and discuss the potential of novel strategies to enhance GvL activity of the transplant.

The competence of cellular immunity depends on a diverse T-cell receptor (TCR) repertoire arising from thymic output. Normal thymopoiesis arises from marrow-derived CD3(-)CD4(-)CD8(-) triple-negative T-cell progenitors (TN), which develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive, DP) transiently, leading to de novo T-cell production. Interleukin-7 (IL7) is a singularly important common γ-chain IL involved in normal thymic development. Our previous work has demonstrated that γc cytokines fused with granulocyte-macrophage colony stimulating factor (GMCSF) at the N-terminus acquire unheralded biological properties. Therefore, to enhance thymopoiesis, we developed a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 leads to cortical thymic hyperplasia including the specific expansion of CD44(int)CD25(-) double-negative 1 (DN1) thymic progenitors. During murine cytomegalovirus (mCMV) infection of aged animals, GIFT7-mediated neo-thymopoiesis led to increased absolute numbers of viral-specific CD8(+) T cell. Our work demonstrated that thymic precursors can be therapeutically repopulated and its reconstitution leads to meaningful central and peripheral T-cell neogenesis, correcting immune dysfunction arising from age-associated thymic atrophy.

Donor T-lymphocytes are effective adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), but life threatening complications related to GVHD limits its clinical application. Recent advancement in the field of immunotherapy has directed our interest to enhancing the anti-tumor response of donor T cells by modulating expression of checkpoint blockade molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and foxp3, the transcription factor associated with regulatory T cells. The two ligands of PD-1, PD-L1 or PD-L2 are highly expressed in the presence of inflammatory signal induced by infection or cancer and PD-1/PD-L1 interaction negatively regulates T-cell antigen receptor (TCR) signaling and dampen T cell cytotoxic activity. Herein, we studied the role of PD-1, CTLA-4 and transcription factor foxp3 expressing donor CD4+ and CD8+ T cells in the development of GVHD. Methods: We have used two established allo-HSCT murine GvHD models. Lethally irradiated wild type (WT) B6, PD-L1 knock out (KO) B6 and PD-L2 KO B6 mice were transplanted with 2 x 106 splenic T cells and 2 x 106 T cell depleted bone marrow (TCD BM) cells from H-2Kdonors. Lethally irradiated CB6F1 recipients were similarly transplanted with splenocytes and TCD BM cells from B6 donors. Acute GvHD scores were determined by combining scores obtained from histological tissue sections and weight-loss, posture, activity, fur texture and skin integrity following standard published procedures. The activation status of donor T-cells and BM and host-derived non-T cells in GvHD target organs was analyzed by flow cytometry. Data from allo-HSCT recipients were compared with the respective data obtained from B6 à B6 syngenic HSCT (syn-HSCT) recipients. Serum cytokines were determined by Luminex assay. Results: PD-L1 KO B6 allo-HSCT recipients had significantly increased acute GvHD scores compared with WT B6 allo-HSCT recipients (p Collectively, these data indicate that severe GvHD occurs in allo-HSCT recipients in spite of increased numbers of PD-1, CTLA-4 and PD-L1 expressing donor and host cells. The occurrence of severe GvHD in these allo-HSCT models systems was associated with markedly reduced levels of CTLA-4 and foxp3 transcription factor expressing Tregs indicating that these pathways may be more relevant to controlling GvHD than PD-1:PD-L1 expression. Disclosures No relevant conflicts of interest to declare.

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

Introduction: Avascular necrosis (AVN) of the femoral head is a devastating disease that often leads to the destruction of the hip. Core decompression of femoral head is the commonest of all conservative surgical treatments where an 8-10 mm cylindrical core of bone is removed from antero-lateral segment of the femoral head. But it is employed only in early stages of the disease and its results are still of controversial efficacy and most of patients ultimately require total hip arthroplasty. Laser irradiation stimulates the reparative process in the bone and marrow while local hyperthermia produced by the laser vaporizes some of necrotic areas leading to a reduction of the intraosseous pressure which is the fulcrum of AVN regardless of the cause. Objectives: The aim of this experimental study was to evaluate the outcome of core decompression by laser osteoperforation for AVN of the femoral head. Subjects and methods: The study was carried out at the Institute of Laser Surgery and Hospital, Dhaka, Bangladesh which included 62 hips with AVN in 40 patients. Preoperative and Postoperative Clinical assessment and Radiological staging were done using ‘Harris Hip Score (HHS)’ and ‘Ficat and Arlet staging’ respectively. Under spinal anesthesia and Fluoroscopic guidance all hips were subjected to ‘Core decompression by Laser Osteo-perforation’ using 970 nm Diode laser through 18G spinal needle via trans-trochanteric and trans-capital approach. Results: All patients were followed up at intervals of three weeks, three months, six months and then yearly (cases with minimum 3 follow ups were included in this study). According to HHS our results are as : 40.3% - excellent; 21.0% - good; 29.0% - fair; 9.7% - poor. No significant side effects or complications were observed so far. Conclusion: Core decompression by laser osteoperforation has been proven so far to be an effective, safe and minimally invasive treatment modality for AVN of the femoral head.

Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1-mediated cellular immunity. We previously reported that VIP-knockout (VIP-KO) mice have enhanced cellular immune responses and increased survival following murine cytomegalovirus (mCMV) infection in C57BL/6 mice. In this study, we tested whether treatment with a VIP receptor antagonistic peptide protects C57BL/6 and BALB/c mice from mCMV-infection. One week of daily subcutaneous injections of VIPhyb was non-toxic and did not alter frequencies of immune cell subsets in non-infected mice. VIPhyb administration to mCMV-infected C57BL/6 and BALB/c mice markedly enhanced survival, viral clearance, and reduced liver and lung pathology compared with saline-treated controls. The numbers of effector/memory CD8+ T-cells and mature NK cells were increased in VIPhyb-treated mice compared with PBS-treated groups. Pharmacological blockade of VIP-receptor binding or genetic blockade of VIP-signaling prevented the up-regulation of PD-L1 and PD-1 expression on DC and activated CD8+ T-cells, respectively, in mCMV-infected mice, and enhanced CD80, CD86, and MHC-II expression on conventional and plasmacytoid DC. VIPhyb-treatment increased type-I IFN synthesis, numbers of IFN-γ- and TNF-α-expressing NK cells and T-cells, and the numbers of mCMV-M45 epitope-peptide-MHC-I tetramer CD8+ T-cells following mCMV infection. VIP-treatment lowered the percentage of Treg cells in spleens compared with PBS-treated WT mice following mCMV infection, while significantly decreasing levels of serum VEGF induced by mCMV-infection. The mice in all treated groups exhibited similar levels of anti-mCMV antibody titers. Short-term administration of a VIP-receptor antagonist represents a novel approach to enhance innate and adaptive cellular immunity in a murine model of CMV infection.

Abstract 3289 Functional T cell immunity depends on the diversity of T cell receptor (TCR) repertoire. Numerous cytolytic assaults can perturb effector cell fitness by inhibiting thymic de novo T cell production or predisposing peripheral CD8+ T cells to exhaustion. In fact, age-associated thymic atrophy and PD-1 upregulation remain the critical components of immune dysfunction. Normal thymopoiesis arises from marrow-derived CD3−CD4−CD8− triple-negative T cell progenitors (TN) which seed the thymic cortex. Thereafter, TN develop into mature single-positive (SP) CD4 or CD8 T cells after expressing both CD4 and CD8 (double-positive-DP) transiently, leading to de novo T cell production. Interleukin-7 (IL7) is a singularly important common γ-chain interleukin involved in this process. In order to pharmacologically enhance thymopoiesis, we report the development of a novel biopharmaceutical based on the fusion of GMCSF and IL7, hereafter GIFT7. Systemic administration of GIFT7 compared to IL7 in young immune competent mice leads to a transient increase in the number of DN1 (5.48±1.01 v.s. 2.74±1.07 × 106, p=0.046) by day 7. Total thymic cellularity significantly increases in GIFT7-treated group by day 14 (237±51.3 v.s. 123±25.6 × 106, p=0.026). GIFT7-mediated hypertrophic effect is significantly more pronounced in aged thymi: 111±21.8, 67.8±13.9, 60.3±4.6 × 106 for 7-dose of (5ug kg−1) GIFT7, IL7, or PBS treatment respectively. We observed a significant increase in DN1 and DN4 subsets with the greatest fold difference in the CD44int DN1 subset. Histologically, GIFT7 administration leads to significant cortical thymic hyperplasia (cortex: medulla ratio 2.67:1). Functionally, GIFT7 enhances viral-specific CTL responsiveness as modeled by murine cytomegalovirus (MCMV) peptide MHC+ tetramer enumeration (10.4±3.8 v.s. 2.9±1.4 × 106 for GIFT7 and untreated respectively, p In the periphery, GIFT7 selectively expand a CD8+ subset with a Central Memory (CM) phenotype defined as CD8+CD44+CD62L+CCR7+KLRG−CD27+PD1−. This unique expansive effect of GIFT7 is also demonstrated on peripheral blood mononuclear cells (PBMC) derived from non-human primates (NHP), in that GIFT7 stimulation of pre-activated NHP-PBMC leads to a two-fold increase in total cell number after 3 days and >80% of Ki67+ expression in both CD4+ and CD8+ compartments. Interestingly, GIFT7-mediated proliferation in CD8+ T cells is accompanied by a 40% decrease in the mean fluorescent intensity (MFI) of PD1 expression. Our work identifies a CD44intCD25− subset of DN thymocytes most responsive to IL7R-mediated STAT5 hypersignalling and their function as T cell progenitors in GIFT7-driven thymic reconstitution. This points to the translational potential of GIFT7 or GIFT7-primed T lineage cells as treatment of immune malfunction. Disclosures: No relevant conflicts of interest to declare.

Abstract 255 Background: Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods: To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results: Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures: No relevant conflicts of interest to declare.

Abstract 1002 Background: Vasoactive intestinal peptide (VIP) is a neuropeptide hormone and type 2 cytokine that inhibits Th1 immunity and induces the generation of regulatory T-cells. We have recently reported that non-transplanted mice “knocked-out” for VIP and syngeneic transplant recipients of VIP-knockout (KO) BM had dramatically improved survival, viral clearance, and increased numbers of specific antiviral CD8+ T-cells following murine cytomegalovirus (mCMV) infection (JI 2011. 187:1057–65). In this study, we used a small molecule VIP antagonist as well as VIP-KO mice to further investigate effects and mechanisms of VIP-signaling on antiviral immune responses in wild type (WT) non-transplanted mice and following allogeneic BMT. Methods: B10BR (CD45.2, H-2Kk) and CB6/J F1 (CD45.2, H-2Kb/d) mice were transplanted with 3 × 103 FACS-sorted hematopoietic stem cells (HSC), 5 × 104 dendritic cells (DC), and 0.3, 1, or 3 × 106 splenic T-cells either from VIP-KO (CD45.2, H-2Kb) or WT donors after myeloablative conditioning (11Gy). WT mice and BMT recipients transplanted with WT grafts were treated with daily subcutaneous injection of VIP antagonist (10 μg/100μL per mouse) or PBS for 7 days (from one day prior to infection to 6 days post-infection). BALB/C mice, B6 VIP-KO and WT littermates, as well as CB6/J F1 BMT recipients, were infected with graded doses (LD10, LD50 and LD90) of mCMV by intraperitoneal injection. Survival, viral load, antigen specific T-cells, and clinical scores of graft versus host disease (GvHD) were assessed at distinct time-points post-BMT or after mCMV infection. The expression of co-stimulatory or co-inhibitory markers (CD25, CD62L, CD69, PD-1, FoxP3, PD-L1, CD80, CD86, and MHC-II) and intracellular expression of cytokines (IL-10, IFN-γ, TNF-α, and IL-12) on T-cells and DC from the mice were measured by flow cytometry. Results: Improved survival was seen in mCMV-infected allogeneic B6→CB6/J F1 transplant recipients of VIP-KO grafts (100%) compared with recipients of WT grafts treated with PBS (40%). Allogeneic recipients of VIP-KO grafts and allogeneic recipients of WT grafts treated with VIP antagonist had increased viral clearance and enhanced in vivo killing of viral-peptide-pulsed targets compared with PBS-treated recipients of WT grafts. No difference in the incidence or severity of acute GvHD was seen in allogeneic BMT recipients of graded doses of VIP-KO versus WT splenic T-cells (0.3, 1, and 3 × 106) in murine MHC mis-matched BMT models. Allogeneic transplant recipients of VIP-KO grafts and WT grafts treated with VIP antagonist, infected with low dose mCMV, had lower levels of PD-L1 and PD-1 expression on DC and T-cells, respectively, and higher levels of CD80, CD86 and MHC-II expression on conventional DC (cDC) and plasmacytoid DC (pDC) compared with recipients of WT allografts treated with PBS. Recipients of VIP-KO grafts and recipients treated with VIP antagonist had higher-levels of IL-12+ cDC, activated CD25+/CD69+ CD4 and CD8 T-cells, and more mCMV-M45-peptide MHC-I tetramer+ CD8+ T-cells compared with recipients of WT grafts treated with PBS. Absence of VIP-signaling led to enhanced intracellular expression of IFN-γ and less IL-10 expression in T-cells from mCMV-infected recipients of VIP-KO B6→CB6/J F1 allogeneic transplants, and mCMV-infected, VIP antagonist-treated recipients of WT allogeneic transplants. In the absence of mCMV infection, the numbers of regulatory T cells (Treg) were similar among VIP-KO mice, WT mice treated with VIP antagonist, and PBS-treated WT controls. In contrast, mCMV-infected VIP-KO mice had significantly fewer Treg compared with mCMV- infected WT mice, non-infected WT mice and non-infected VIP-KO mice. Conclusion: Genetic or pharmacological blockade of VIP-signaling enhanced both innate and adaptive antiviral immune responses in allogeneic BMT recipients without significantly elevating GvHD. Selective targeting of VIP-signaling represents a novel therapeutic approach to enhance antiviral immunity in the setting of immunodeficiency and allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients. Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures: No relevant conflicts of interest to declare.

Clinical studies suggested an association between thyroid cancer outcome and patient immunologic function. Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) and Programmed Death 1 (PD-1) are strong negative regulators of immune function whose aberrant function has been associated with poor outcomes in patients with solid malignancies. We carried out this study to compare the degree and patterns of expression of CTLA4 and PD-1 in peripheral circulating mononuclear cells in adult patients with thyroid cancer (N=21) compared with adult healthy controls (N=5). We used multiparameter flow cytometry to determine the absolute cell count per ml of blood for the cells of interest. Functional assays of immunologic competence of B-cell, T-cell as well as NK cell subpopulations was assessed by granzyme-B and interferon (IFN) gamma production following PMA/ionomycin stimulation in vitro using standard assay method. Differences in specific cell count between patients and healthy control was assessed by F-test statistics and p-value There was no significant difference between cancer patients and healthy controls in the overall numbers of T-cell, B-cell, and NK cell subsets. Similarly, the proportion of CD4, CD8 and NK cell subsets with CTLA4, PD-1, IFN-gamma or Granzyme-B expression with and without PMA/ionomycin stimulation were not significantly different when comparing patients with thyroid cancer to healthy controls. Within the thyroid cancer patient population, however, the distribution of CD4 (+) T-cells, total B-cells, plasma cells, and PD ligand 1-expressing monocytes, macrophages and dendritic cells i.e. CD64 (+) PD-L1 (+) was significantly different between patient with active disease and those in remission. The Granzyme-B (+) CD4 (+) T-cell was significantly lower in patients in remission than in patients with active disease both with and without in vitro activation with PMA/ionomycin (0.08 vs. 0.19 per ml blood × 10⁁6; p=0.03). Intriguingly, functional profiling in a limited number of thyroid cancer patients revealed a markedly impaired ability of CD8 (+) T-cells to produce IFN-gamma and Granzyme B following PMA/ionomycin stimulation. Thus suggesting possible immune exhaustion in this subpopulation of thyroid cancer patients. Overall, our results show a trend toward impaired cytotoxic immune pathway in thyroid cancer patients with active disease burden when compared to patients in remission. A similar trend was observed in comparison to healthy subjects but the small number of control subjects limits statistical significance. Further elucidation of this finding using larger patient sample is clearly warranted and currently ongoing. (Supported by Career Development Award (NCI P50 CA128613) to TK Owonikoko and the Georgia Cancer Coalition Distinguished Scholar Award to DM Shin, SS Ramalingam, FR Khuri, and TK Owonikoko) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2011-5525

Abstract 3561 Poster Board III-498 Background Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Immunosuppressive drugs limit clinical GvHD but increase relapse and susceptibility to opportunistic infections and also result in drug related toxicities. To develop an alternative approach to control GvHD, we tested the immunomodulatory immune properties of flagellin, a bacterial protein that agonistically binds with TLR5 and protects mice from radiation-induced gut injury, in murine allo-BMT models. Methods We used established BA.B10 (H-2K) → C57BL/6 (H-2b) MHC mismatched experimental models of allogeneic HSCT in which GvHD is a major complication. 50 μg LPS-free purified flagellin in PBS or PBS alone were administered intraperitoneally in two doses: 3 hours before fractionated irradiation (5.5Gy X 2 fractions) and 1 day post-transplant. Allografts were performed 1 day after irradiation and contained 5 million (M) T-cell depleted bone marrow (BM) cells and 5 M plastic non-adherent splenocytes from naïve BA.B10 donors. The primary end-points was survival; HSCT recipients were monitored twice a day for mortality and GvHD signs and recipients having more than 25% weight loss were sacrificed. Blood, spleen, thymus and BM were collected from surviving mice on day 132 post transplant, live cells counted, and immune phenotypes were analyzed by FACS. The numbers and phenotype of immune cells in organs from flagellin-treated HSCT recipients were compared to the similar immune cells per organs analyzed from a normal B6 mouse having similar age of HSCT recipients. Results Flagellin treated recipients had 15% weight-loss and 33% transplant-related death by 132 days post transplant versus severe acute GvHD and 100% early post-transplant mortality among control HSCT recipients that received PBS. Flagellin-treated recipients had 100% donor chimerism with limited clinical signs of GvHD. While total cell numbers per spleen (8.2 ± 5.4M) and thymus (7.1 ± 4.9M) were very low in flagellin-treated recipients compared to normal B6 mice (>100M/organ), the cell numbers isolated from blood (8.9 ± 2.6 M/ml) and BM (104.5 ± 37.4 M) were similar to non-transplanted B6 mice (11.4M/ml blood and 108 M/BM, respectively). BM of flagellin-treated HSCT recipients contained similar numbers of CD4+ T cells (4.6 ± 2.7 M) and CD8+ T cells (2.5 ± 1.4 M) as normal B6 mice (4.03M and 1.3M, respectively). Numbers of naïve and memory CD4+ T-cells in the BM were similar between flagellin-treated and control mice: CD4+CD62L+(0.7 ± 0.2 versus 0.5M); CD4+CD62L- (3.9 ± 2.5 versus 3.5 M); CD4+ CD44hi (2.8 ± 1.4 versus B6 3.6M); and CD44lo (1.7 ± 1.3 M versus 0.44M). In contrast, flagellin-treated HSCT recipients had more naïve CD8+ T-cells but similar memory CD8+ T-cells in their BM compared with control mice: CD8+CD62L+(2.6 ± 1.4 versus 1.0M); CD8+CD62L- (1.7 ± 1.2M versus 0.3 M); CD8+CD44hi(0.8 ± 1.1 versus 0.7M); and CD8+CD44lo (0.7 ± 0.3M versus 0.6 M). The numbers of total CD3+ T cells, NK cells, and lin-CD11b-Sca-1+Ckit+ Stem cells in the BM were also similar comparing flagellin-treated recipients with non-transplanted B6 control mice. The number of CD3-B220+ B cells in the BM were lower in flagellin-treated recipients compared to B6 mouse (18.1 ± 3.2M versus 43.1M) as were the numbers of T-cells and B-cells per mL blood of flagellin-treated mice were found lower compared with the blood of normal B6 mouse: 0.8 ± 0.2M T-cells/mL versus 2.1M/mL; 5.5 ± 2.5M B cells/mL versus 9.1M/mL. Although the cellularity of the thymus in flagellin-treated animals was very low compared to normal B6 mice, a usual percentage (62.5 ± 10.5%) of thymocytes were of CD4/CD8 double positive, indicating functional thymopoiesis in these recipients. Conclusion Flagellin protected allogeneic HSCT recipients from irradiation-induced BM damage and prevented lethal GvHD in a major MHC mis-matched model of GvHD. Flagellin and other TLR5 agonists may be novel therapeutic approaches to prevent or reduce GvHD in allogeneic HSCT recipients. Disclosures: No relevant conflicts of interest to declare.

Abstract 1165 Poster Board I-187 Background Opportunistic CMV infections remain a major cause of morbidity and mortality in allogeneic hematopoetic stem cell transplant (HSCT) recipients, with prolonged periods of viremia associated with cytopenia and life-threatening end-organ infections. Although CMV infection in allogeneic HSCT recipients may be controlled using anti-viral drugs, re-occurrence of viremia when drug therapy is stopped is common, and the emergence of drug resistant viral strains may render the anti-viral agents ineffective. The lack of functional anti-viral immunity that leads to CMV reactivation in immunocompromized HSCT recipients is a complex phenomena and may be due to 1) failure or insufficient donor/host innate immunity to prevent reactivation of CMV from their latent state, 2) failure to effectively process and present CMV antigens by the appropriate APCs to anti-viral T cells due to conditioning regimens used to prevent graft rejection and graft-vs-host disease (GvHD); 3) lack of response of CMV specific memory T cells to control CMV infection. Lack of any or all of these three elements of immune responses are hypothesized to be responsible for clinical CMV reactivation or infections in allogeneic transplant recipients. Determining which of these three elements of effective anti-viral immunity are most critical to preventing CMV infection would help guide novel therapies designed to prevent or treat opportunistic viral infections of high-risk patients. Method Emory is the core immune-monitoring laboratory for multi-center clinical study CTN 0201. As part of this study, we have samples from 69 HLA-A*2+ patients who received myeloablative conditioning and allogeneic HSCT from unrelated donors. HLA-A*2 restricted HCMV peptide pp65 (495-503, NLVPMVATV) specific tetramer+ CD8+ T-cells were determined from the blood samples obtained on 3, 6, and 12 months post transplant. Blood samples from three patients (2 HLA-A*2+ and 1 HLA-B*7+) treated at Emory had simultaneous data on both CMV viral load (measured by RT PCR) and the anti-CMV specific T-cells using the HLA-A*2 and HLA-B*7 [CMV pp65(NLVPMVATV)] tetramer reagents. Results Out of 69 patients, 5% had more than 1% CD8+ T-cells expressing the HLA-A*2 restricted CMV pp65 specific T-cell receptor at 3 months post-transplant, with an increasing proportion of patients showing the presence of at least 1% CMV-specific T-cells at 6 and 12 months post-transplant. Serial simultaneous measurements of CMV viral load and anti-CMV-specific T-cells are shown in Table 1 for 3 pts treated at Emory. In each case, evidence of active CMV replication was accompanied by a rapid expansion of CMV pp65-specific T-cells in the blood. Contraction of the CMV-specific T-cell compartment subsequently occurred, in conjunction with antiviral therapy and successful clearance of the virus. CMV reactivation events were not associated with major changes in the numbers of CD4+ T-cells, monocytes, or granulocytes. Conclusions Successful control of CMV reactivation is associated with a rapid and specific CD8-mediated immune response, which works in conjunction with antiviral therapy to limit viral replication. We would predict that the inability to control CMV reactivation, which is observed in a subset of pts following allogeneic transplantation, may not be associated with the same degree of target-specific immune expansion. This may be due to low absolute T-cell numbers, lack of innate immune responses or suppression of the ability of memory T cells to become effector T cells. Ongoing analyses will correlate measurements of tetramer+ T-cells in the 0201 dataset with laboratory evidence for CMV viremia and clinical infection. Disclosures No relevant conflicts of interest to declare.

Abstract 2458 Poster Board II-435 Introduction: When used as therapy for hematopoietic malignances, allogeneic hematopoietic stem cell transplantation (HCT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host-disease (GVHD) is a potentially lethal complication of allogeneic BMT. Thus, inhibition of GVHD, while preserving GVL and protective responses against infectious agents, can enhance the therapeutic potential of BMT. GVHD is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues. Protein kinase C theta isoform (PKCθ) is crucial in TCR signaling and regulates the nature of lymphocyte-specific effector responses. The aim of this study was to investigate the significance of PKCθ in donor T-cell-mediated GVHD, anti-leukemia, and antiviral infection. Methods: Our results were validated using clinically relevant murine bone marrow transplantation (BMT) models. GVHD was measured by recipient survival and clinical signs such as body weight loss. Leukemia/lymphoma was induced by intravenous injection of a luciferase expressing A20 clone (a B lymphoma cell line derived from BALB/c mice). Tumor invasion was quantitated using the Xenogen IVIS-200 bioluminescence imaging system. Murine cytomegalovirus (MCMV) infection was induced by intraperitoneally injecting MCMV in the recipients after GVHD was established. Results: Using 3 distinct mouse models of allogeneic BMT, we found that T cells lacking PKCθ are unable to undergo robust expansion and cause damage to recipient hematopoietic or epithelial tissues, demonstrating that an essential requirement for PKCθ in alloreactivity and GVHD development. In contrast, PKCθ-/- T cells retain the ability to respond to virus infection post-BMT. Mechanistically, absence of PKCθ raises the threshold for T cell activation that selectively impacts alloresponses, but T cell responses triggered by infection or following administration of antigen plus microbial adjuvant are relatively well preserved in the absence of PKCθ Furthermore, we evaluated the role of PKCθ in GVHD and GVL responses, and found that PKCθ-/- T cells have at least a 10-fold reduction in the ability to induce GVHD but only a ∼2.5-fold reduction in GVL compared to WT T cells. Thus, absence of PKCθ impacts GVHD responses more significantly than GVL responses. Conclusion: These findings validate PKCθ as a potentially unique therapeutic target that is required for GVHD induction but not for GVL or protective responses to infectious agents in allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

We are investigating methods to reduce the graft-versus-host disease (GVHD) potential of donor T-cells while retaining graft-versus-leukemia (GVL) activity in allogeneic HSCT. Previous investigations by our group and others in have shown that naive CD4 T-cells induce severe acute GVHD, while memory CD4 T-cells do not induce GVHD but retain GVL activity in murine transplant models. These findings have led to studies for the development of methods to increase the number of memory T-cells available for transplant. The calcium ionophore, ionomycin, is a T-cell activating agent and mitogen. By increasing intracellular Ca2+ levels, ionomycin is induces T-cell activation through signaling mechanisms including phospholipase C activation, hydrolysis of phosphoinositides, and activation of protein kinase C. Differences in memory and naive T-cell responses to ionomycin have been attributed to resistance of memory T-cells to increases in Ca2+. Memory T-cells lack intracellular Ca2+ stores, and are also resistant to influx of Ca2+. Brief low dose ionomycin exposure (20min, 2μM) of T-cells, leading to increased density of naive T-cells, has previously been exploited as a method for separating memory and naive T-cells by Percoll gradient separation. Since ionomycin exposure induces T-cell activation through native Ca2+ dependent signaling mechanisms, we hypothesized that ionomycin-treated T-cells would shift to an activated/memory T-cell phenotype. Murine splenic T-cells were treated with 1.3μM ionomycin for 4hr. Memory and naive T-cell subsets and activation markers were analyzed by flow cytometry. 75% and 85% of untreated CD4 and CD8 T-cells, respectively, had the CD62L+ naive phenotype. These numbers were dramatically reduced to 7% and 17% after ionomycin exposure, representing a shift to the memory T-cell phenotype. Viability of T-cells was not significantly affected. The majority of remaining CD62L+ naive T-cells expressed activation markers CD25 and CD69. The fraction of CD4+CD25+Foxp3+ regulatory T-cells was also determined by intracellular staining of the transcription factor and co-expression of surface markers. CD4+CD25+Foxp3+ regulatory T-cells represented 4% of untreated CD4 T-cells and 3% of ionomycin-treated CD4 T-cells. While ionomycin has been used for many years in studies of T-cell activation, to our knowledge this is the first demonstration of a rapidly-induced shift of naive T-cells to a memory phenotype. A pilot experiment was conducted testing the GVHD activity of ionomycin-treated splenocytes (SP) in B6→ (B6 × Balb/C)CB6F1 recipients. 5 × 106 T-cell depleted bone marrow cells (TCD-BM) were transplanted along with 10 × 106 treated or untreated SP. Mice that received untreated SP all died from acute GvHD by 34 days after transplant, while all recipients of ionomycin-treated SP survived until the experiement was terminated at day 49 (average weight loss was 25%, data not shown). Continuing experients will refine the dose to further reduce GVHD symptoms and also test GVL activity of the treated cells. Treatment of donor T-cells with ionomycin may represent a clinically applicaple method to engineer donor lymphocyte infusions that are safer for HSCT patients. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49. Figure 1. Survival of CB6F1 recipients after transplant with 5 million B6 TCD-BM and 10 million B6 splenocytes that were either untreated or stimulated ex-vivo with a combination of PMA, ionomycin and brefeldin-A for 4 hours. 5 recipient animals per group. The experiment was terminated at day 49.

Background: Allo-reactive donor T cells are primarily responsible for GvHD in allogeneic BMT. A number of studies have shown that increased allo-reactivity is found among the CD62L+ subset of donor T-cells, but the mechanisms for organ specific allo-reactivity are poorly defined. Our hypothesis is that rapid proliferation and migration of CD62L+ naive donor CD4+ and CD8+ T cells to specific organs leads to acute GvHD. Methods: We used a parent (C57BL/6) to (C57BL/6 × BALB/c) CB6F1 allogeneic BMT model with a combination of T cell depleted BM (TCD BM) and splenocytes. 30 × 106 congeneic donor splenocytes labeled with CFSE were transplanted with 5 × 106 TCD congeneic BM into lethally irradiated (11Gy) CB6F1 mice. Recipients were sacrificed within 3.5 days of transplant and FACS was used to measure proliferation of CFSE-labeled donor T-cells isolated from blood, spleen, liver, lungs, thymus, BM, lymph nodes, and peritoneal exudates cells (PEC). Syngeneic C57BL/6 recipients served as controls. At least 5 mice per group were used in each experiment. Results: There was increased homing of CFSE-labeled donor T-cells to most organs in allogeneic compared to syngeneic BMT recipients. CD45.1+ donor cells were 4-fold higher in spleen, p=0.01; 9-fold higher in liver, p=0.002; 14-fold higher in PEC, p=0.017; 136-fold higher in lung, p=0.0006; 126-fold higher in BM, P=0.002, 1482-fold higher in thymus p=0.002 compared to syngeneic recipients. Allogeneic and syngeneic recipients had equivalent numbers of donor CFSE-labeled lymphocytes in PBMC and lymph nodes. The tissue specific homing of CD4+ and CD8+ donor T-cells was also found significantly higher in most organs except the PBMC and LNs. Donor splenocytes were 80% CD62L+ before transplant, but the frequency of CD62L+ donor T-cells had declined to 15–16% in BM, 4–10% in liver, 17–30% in spleen and 10 to 25% in the thymus within 3.5 days post-transplant. In syngeneic recipients, 80% of donor T-cells remained CD62L+ within 3.5 days post-transplant. Most donor T-cells that divided rapidly lost expression of CD62L, while non-replicating donor CD4+ and CD8+ T cells remained predominately CD62L+. The expression of CD44 on donor T-cells were the opposite, with CD44+ cells undergoing less, and CD44− cells dividing more in allogeneic transplant recipients. In syngeneic BMT, donor CD4+ and CD8+ T-cells underwent minimal proliferation within the first 3.5 days post-transplant. Intracellular cytokine staining showed that high levels of IFN-g and TNF-a synthesis was seen among CD62L+ CD4+ and CD8+ T cells that had yet to divide (and had un-diluted CFSE staining). Conclusion: Migration of allogeneic donor T cells to tissues and local proliferation occurs rapidly after allogeneic BMT compared to recipients of syngeneic transplants. The dissociation of CD62L expression from lymph node homing suggests lack of the CD62L-receptor expression in lymph node HEV following irradiation, or a dominant effect of other chemokine receptors in directing donor T-cell preferentially to other organs. The marked and preferential homing of donor T-cells to the recipient thymus and bone marrow may play a role in achieving donor hematopoietic and T-cell chimerism in recipients of allogeneic BMT. Tissue specific homing of naive CD62L+ donor T-cells, with a high proliferative capacity, is likely responsible for the initiation of acute GvHD at these sites.