Immune Suppressive Drugs Research Articles

Efficacy and Safety of the BRAF Inhibitor Vemurafenib in Hairy Cell Leukemia Patients Refractory to or Relapsed after Purine Analogs: A Phase-2 Italian Clinical Trial

▪BACKGROUND AND AIMS: Hairy cell leukemia (HCL) is very sensitive to purine analogs (PAs), but ~40% of patients relapse and become progressively less responsive to these myelotoxic and immune-suppressive drugs. Having discovered the BRAF-V600E kinase-activating mutation as the genetic lesion underlying HCL (Tiacci et al, NEJM 2011;364:2305), we performed the first clinical trial of a BRAF inhibitor (vemurafenib) in refractory/relapsed HCL. In particular, this is a phase-2, academic, single-arm, Italian, multi-center (n=8) study (HCL-PG01; EudraCT 2011-005487-13).METHODS: In 11 months we enrolled 28 BRAF-V600E+ HCL patients, needing therapy due to cytopenias and including: i) 6 patients primary refractory to a PA; ii) 21 patients who relapsed early and/or repeatedly after PAs and had received a median of 4 previous therapies; and iii) a 81-year old patient showing severe myelotoxicicity after a PA (discouraging its further use). Previous treatments other than PAs included interferon, rituximab and splenectomy in 12, 14 and 8 patients, respectively. Complete remission (CR) required resolution of cytopenias (N≥1500/mmc, PLT≥100000/mmc, Hb≥11 g/dl), no morphological evidence of HCL cells in the bone marrow biopsy and blood smear, and no splenomegaly. Partial remission (PR) required resolution of cytopenias, and a ≥50% reduction of splenomegaly and of marrow and blood HCL involvement by immunophenotyping. Two patients were not evaluable as they went off-study after ≤1 week of treatment (due to drug-unrelated acute myocardial infarction and consent withdrawal after grade-3 drug-related reversible pancreatitis).RESULTS: Vemurafenib, given orally at the dose of 960 mg twice daily on an outpatient basis for a median of 16 weeks, was generally well tolerated. Drug-related adverse events (mainly arthralgias, skin toxicities, pancreatitis; no myelosuppression) were frequent, but reversible in all patients, and were typically grade 1-2. Only 7 patients developed grade 3 events, and none grade 4 events. Although we did not observe any cutaneous squamous cell carcinomas/keratoachantomas (as reported in BRAF-V600E+ melanoma patients treated with vemurafenib), 3 patients developed 2 basaliomas and 1 superficial melanoma, all treated with a simple excision.Notably, overall response rate was 96% (25/26 patients): 9/26 (34.6%) CRs and 16/26 (61.4%) PRs, obtained after a median of 8 and 9 weeks respectively. CR and PR patients included 1 and 5 primary refractory ones, respectively, as well as 4 and 10 not responding to the last prior treatment, respectively.In all CR patients immunohistochemistry showed minimal residual disease (≤10%) at the end of treatment. Six of 9 (67%) CR patients enjoyed normal blood counts at a median of 13 (range 12-15) months from the end of treatment (see Figure): 3 of these 6 patients showed no morphological evidence of HCL in the bone marrow biopsy (complying with a continuous CR) at 12, 13 and 15 months, respectively, whereas the other 3 lost the bone marrow CR status, all at 12 months. The remaining 3/9 CR patients (33%) developed a mild cytopenia (N ~1000/mmc or PLT ~80000/mmc) 5, 9 and 12 months post-treatment, respectively: in the 2nd patient the cytopenia remained stable until the last follow-up at 15 months, whereas in the other two cases it worsened requiring therapy 9 and 18 months post-treatment, respectively (see Figure). These two latter patients were recently retreated with vemurafenib for 12 and 4 weeks, and obtained a PR and a second CR.Among the 16 PR patients, 5 (31%) mantain normal blood counts at a median of 12 (range 8-17) months post-treatment (see Figure). The other 11 PR patients developed cytopenia(s) after 3 months of median follow-up (range 5-10): in 6 patients (38%) no anti-leukemic therapy was started at a median of 9 (range 6-12) months post-treatment, whereas in the remaining 5 cases (31%) cytopenia(s) worsened requiring therapy at a median of 8 (range 5-11) months of follow-up (see Figure). Four of these latter 5 patients were retreated with vemurafenib for 12 weeks: 3 cases had a minor response and the last one witnessed a second PR that lasted less than the first PR (3 versus 9 months).CONCLUSIONS: In heavily pre-treated HCL patients, a short oral course of vemurafenib was safe, and proved quickly and highly active. Retreatment with vemurafenib was able to reinduce remissions in patients relapsing after a CR, but was less effective in patients relapsing after a PR. [Display omitted] DisclosuresOff Label Use: Off-label use of vemurafenib in hairy cell leukemia will be discussed as part of a clinical research protocol..

Relationship of Epidermal Growth Factor to Cyclosporine-Assocated Magnesium Wasting and Clinical Outcomes Post-Allogeneic Hematopoietic Cell Transplantation

Background: Hypomagnesemia is a frequent complication of allogeneic hematopoietic cell transplantation (HCT) and is related to cyclosporine-induced renal magnesium (Mg) loss. The importance of epidermal growth factor (EGF) in renal Mg reabsorption was suggested after identification of EGF mutation in isolated autosomal recessive hypomagnesemia (IRH). Mg wasting is also associated with EGFR tyrosine kinase inhibitors used in oncologic treatment. Recently, a link between EGF, Mg, and cyclosporine (CSA) in renal transplant recipients has been proposed through downregulation of EGF leading to inhibition of the Mg channel TRMP6 in the distal convoluted tubule, but similar associations have not been investigated post-allogeneic HCT. We hypothesized that lower serum EGF would be associated with higher CSA levels, acute graft versus host disease (GVHD) status, and more severe Mg wasting after allogeneic HCT. Patients and Methods: 107 adult allogeneic HCT recipients from the University of Minnesota enrolled on an immune monitoring study had EGF levels measured by magnetic bead array at day +100 post-allogeneic HCT. Clinical variables collected for analysis included patient age, sex, underlying diagnosis, donor type, HCT conditioning regimen, maximum acute GVHD overall grade and organ involvement prior to day +100, use of oral and intravenous supplemental Mg including cumulative requirements over 2 weeks, and concurrent immunosuppressive drug therapy including cyclosporine (N=66), tacrolimus (N=13), mycophenolate mofetil (N=5), sirolimus (N=22), and prednisone (N=40). Lab variables analyzed included serum creatinine and estimated glomerular filtration rate (GFR), immune suppressive drug levels, Mg levels, and lymphocyte subsets including absolute counts and percentages of NK cells, B cells, CD4+ T cells, CD8+ T cells, and regulatory T cells. Results: 54 patients had acute GVHD (grade I-II n= 34, grade III-IV n =20) and 53 patients did not have acute GVHD prior to the day +100 serum sample collection. The median serum EGF in this cohort was 101 pg/mL (not detectable in 5 samples). Levels of EGF at day +100 showed no relationship with age, underlying diagnosis, donor type, or conditioning intensity. EGF was significantly lower in patients who had acute GVHD grade III-IV prior to day +100 (Figure 1) and nearly 3-fold lower in those with upper GI GVHD involvement versus no upper GI GVHD involvement (median 43 pg/mL versus 119 pg/ml, p=0.007). Serum EGF negatively correlated with CSA level (Spearman's rho = -0.34, p=0.007), but not with tacrolimus levels, sirolimus levels, prednisone dose, or serum creatinine. Mg requirements, measured as either daily dose or cumulative dose over 2 weeks prior to the EGF sampling, were not associated with serum EGF overall, but those with EGF > 100 pg/mL showed higher magnesium requirements (average daily dose (1060 ± 142 mg/day versus 600 ± 85 mg mg/day, p=0.02) and average cumulative dose over 2 weeks (14.6 ± 2 grams versus 9 ± 1.3 grams, p=0.04) prior to the blood draw. Patients with or without acute GVHD required similar Mg supplementation. EGF levels showed a modest correlation with overall lymphocyte percentage in the peripheral blood (Spearman's rho = 0.24, p=0.01), but not with specific lymphocyte subsets at day +100. Serum EGF levels > 100 pg/mL at day +100 were associated with improved 2-year survival (RR 0.46, 95% CI 0.22–0.94, p=0.03, Figure 2). Conclusions: Patients with prior grade III-IV acute GVHD, and in particular upper GI GVHD, had lower EGF levels at day +100. This may be due in part to decreased EGF production from Brunner's glands in the duodenum versus excess EGF losses. The inverse relationship between CSA and EGF may be direct or may represent patients with more severe acute GVHD being maintained with higher drug levels. Serum EGF was negatively correlated with CSA levels but not Mg requirements or serum creatinine/estimated GFR. While studies in renal transplantation identified a connection between urinary EGF, Mg wasting, and diminished renal function, circulating levels of EGF in allogeneic HCT did not show a similar correlation. Differences in EGF post-allogeneic HCT are predominantly due to grade III-IV GVHD status, although a potential contribution from CSA effect on the kidney cannot be ruled out. The role of circulating EGF in the long-term outcomes of allogeneic HCT should be further studied. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

The Prevalence of Pneumocystis jiroveci in Bronchoalveolar Lavage Specimens of Lung Transplant Recipients Examined by the Nested PCR.

Background:The use of immune suppressive drugs for organ transplant recipients predisposes them to opportunistic infections, especially by fungal agents. Pneumocystis jiroveci, as an opportunistic pathogen, endangers the patients’ life in those with immune system disorders. Early detection of latent Pneumocystis infection in susceptible patients may help choose the optimal treatment for these patients.Objectives:The aim of this study was to identify and determine the colonization of latent P. jiroveci infection among lung transplant recipients.Patients and Methods:This cross-sectional descriptive study was conducted on lung transplant recipients. Bronchoalveolar lavage (BAL) specimens were collected from 32 patients undergoing bronchoscopy. The samples were aseptically homogenized by 10 mM dithiothreitol, and their DNA was extracted. The mtLSUrRNA gene of P. jiroveci was amplified using nested PCR in two stages. Nested PCR was performed using external primers of pAZ-102-E and pAZ102-H followed by using the PCR product of the first stage and internal primers of pAZ-102-E and pAZ102-L2.Results:The genome of P. jiroveci was revealed by a 346 bp PCR product in the initial amplification and a 120 bp product in the nested PCR. The results showed that seven BAL specimens (21.9%) from lung transplant recipients were positive for P. jiroveci.Conclusions:In molecular epidemiology studies, nested PCR has higher sensitivity than PCR. Results of this study support the colonization of P. jiroveci in patients receiving lung transplantation. Patients who are carriers of P. jiroveci are at a higher risk of P. jiroveci pneumonia.

Recombinant probiotics with antimicrobial peptides: a dual strategy to improve immune response in immunocompromised patients

Bacterial infectious diseases are currently a serious health problem, especially in patients compromised by illness or those receiving immune-suppressant drugs. In this context, it is not only essential to improve the understanding of infectious mechanisms and host response but also to discover novel therapies with extreme urgency. Probiotics and antimicrobial peptides are also favorably viewed as novel strategies in the control of resistant bacteria. The present review will shed some light on the use of probiotic microorganisms expressing antimicrobial peptides as a dual therapy to control bacterial infectious diseases.

Synergic silencing of costimulatory molecules prevents cardiac allograft rejection

BackgroundWhile substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival.MethodVectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed.ResultAllogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10–16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4 + CD25 + FoxP3+ regulatory T cell differentiation in vitro.ConclusionThis study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.

Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A.

Excessive or aberrant generation of neutrophil extracellular traps (NETs) has recently become implicated in the underlying aetiology of a number of human pathologies including preeclampsia, systemic lupus erythromatosus, rheumatoid arthritis, auto-antibody induced small vessel vasculitis, coagulopathies such as deep vein thrombosis or pulmonary complications. These results imply that effective pharmacological therapeutic strategies will need to be developed to counter overt NETosis in these and other inflammatory disorders. As calcium flux is implicated in the generation of reactive oxygen species and histone citrullination, two key events in NETosis, we analysed the roles of both extra- and intracellular calcium pools and their modulation by pharmacological agents in the NETotic process in detail. Interleukin-8 (IL-8) was used as a physiological stimulus of NETosis. Our data demonstrate that efficient induction of NETosis requires mobilisation of both extracellular and intracellular calcium pools. Since modulation of the calcineurin pathway by cyclosporine A has been described in neutrophils, we investigated its influence on NETosis. Our data indicate that IL-8 induced NETosis is reduced by ascomycin and cyclosporine A, antagonists of the calcineurin pathway, but not following treatment with rapamycin, which utilizes the mTOR pathway. The action of the G protein coupled receptor phospholipase C pathway appears to be essential for the induction of NETs by IL-8, as NETosis was diminished by treatment with either pertussis toxin, a G-protein inhibitor, the phospholipase C inhibitor, U73122, or staurosporine, an inhibitor of protein kinase C. The data regarding the calcineurin antagonists, ascomycin and cyclosporine A, open the possibility to therapeutically supress or modulate NETosis. They also provide new insight into the mechanism whereby such immune suppressive drugs render transplant patients susceptible to opportunistic fungal infections.

Utility of immune monitoring in heart transplant recipients on everolimus‐based immune suppression

Everolimus provides effective immune suppression (IS) after heart transplant (HTx). Its pharmacologic properties differentiate everolimus from other IS drugs. A non-invasive immune monitoring (IM) assay test appears to predict the immune state in HTx recipients on standard calcineurin-inhibitor-based IS. The utility of IM in HTx recipients on everolimus-based IS was evaluated. Between June 2005 and June 2011, 34 adult HTx recipients followed up at our center received everolimus and had 381 IM assays that were performed at sixmonths to 16-yr post-transplant. Results of the IM assay were correlated with infection and rejection episodes that occurred during the IM testing. In the everolimus-based IS group, there were 18 infectious episodes and four rejection episodes. The average IM score was significantly lower during infection than at steady state (188±122 vs. 338±137ng/mL ATP, p<0.001) and not significantly different during rejection when compared with steady state (430±132 vs. 338±137ng/mL ATP, p=0.5). The non-invasive IM assay predicts infectious risk in HTx recipients on everolimus-based IS. Its inconclusive association with rejection was probably due to the small number of rejections. Serial longitudinal IM may allow proper adjustment of everolimus doses.

What Is Suppression of Anti–Adeno-Associated Virus Capsid T-Cells Achieving?

In general, recombinant adeno-associated virus (rAAV) vectors so far have been found to be safe and persistent. However, anticapsid immune responses have been observed in various clinical trials. Specifically, these are the development of neutralizing antibody responses, which may interfere with readministration and the development of anti-AAV capsid responses. Based on data arising from a number of rAAV factor IX clinical trials (Manno et al., 2006; Nathwani et al., 2011), a correlation has been made between effector T-cell responses to AAV capsid epitopes and the loss of cells that have been transduced in vivo. Particular emphasis has been placed on the presence of AAV capsid epitope-specific IFN-gamma ELISPOT results in peripheral blood. On the basis of this correlation, a number of investigators have included either prospective immune suppressive drug regimens or therapy with corticosteroids in the posttransduction period into their clinical trials of rAAV vectors. It has been suggested that the effectiveness of such facilitating therapies would be deemed successful initially by abrogation of anticapsid ELISPOT results. The results presented in an article in this issue of Human Gene Therapy by Ferreira et al. (2014) call this reliance on IFN-gamma ELISPOTs into question and raise the broader issue of whether pharmacologic suppression of effector T-cell responses is always necessary. In fact, the report published here was the basis for the licensure of intramuscular (IM) rAAV1-Lipoprotein Lipase (Glybera), the first human gene therapy licensed in the Western world, an agent whose European Medicines Agency (EMA) label indicates a need for immune suppression. In this report, despite the use of immune suppression, 4/5 subjects had positive IFN-gamma ELISPOT to AAV1 capsid epitopes and more significantly had CD8+ cellular infiltrates at the site of injection. However, the systemic and local immune responses did not seem to affect transgene expression or pose a safety risk. It is vital in considering this report to keep in mind that the correlation of anticapsid T-cell responses with transduced cell loss has only been observed clinically when the liver is targeted, as in the two hemophilia programs mentioned above. In fact, the only other IM rAAV1 product for which there is significant immune response data, the rAAV1-alpha-1 antitrypsin (AAT) vector product, has included a series of trials performed without immune suppression. Two successive studies have demonstrated that vector expression persists for at least a year despite effector T-cell responses (Brantly et al., 2009; Flotte et al., 2011). Perhaps more informative is the recent finding that rAAV1-AAT elicits an AAV capsid-specific regulatory T-cell (Treg) response (Mueller et al., 2013), which was documented both in situ and in the peripheral blood. In that study there was also evidence of PD-1 T-cells at the injection sites, indicating that very long-term capsid epitope persistence may have led to effector T-cell exhaustion, which contributed to the development of the robust Treg response. In the current AAV1-lipoprotein lipase study by Ferreira et al., the muscle biopsies also stained positive for FOXP3+ CD4 T-cells. Thus, we believe that positive anticapsid ELISPOTs may be better interpreted in the context of this Treg response. The abundance of Tregs in the rAAV1-AAT study (10% of all infiltrating T-cells in the injected muscle) would suggest that the Tregs are present in sufficient numbers to locally suppress any effector response that might have eliminated transduced myofibers. Taken together, the findings in the study published here, along with the other IM rAAV1 clinical immunology data, suggest that IFN-gamma ELISPOTs do not predict loss of vector expression and do not serve as a reliable indicator of the need for immune suppression. Further studies are clearly needed to determine whether IFN-gamma ELISPOTs are predictive of undesirable elimination of vector-mediated expression with other rAAV serotypes and other routes of delivery. Until then, given the obvious risks associated with immune suppression, it seems prudent not to rely on ELISPOTs alone as the indication for immune suppression, but rather to seek first to correlate such laboratory findings with actual limitations on vector performance.

Fascinating Interaction between Host and Pathogen

Clinical manifestations of disease depend upon host's immune response that is induced by pathogen and modified by the host's innate and adaptive immunity. Immunocompetent children of similar age and nutrition evoke different responses to the same pathogen varying from benign to potentially fatal condition. This results in diverse clinical presentations of a disease, that is different from the standard expected pattern and thus, poses a diagnostic challenge. Even, subsequent progression of a disease is also variable. It is the balance between immune stimulation, immune suppression and immune tolerance that decides the outcome. In case of balanced response, child recovers completely without any damage. However at times, cure is at the expense of permanent sequalae while in case of unfavourable immune response, survival may not be certain inspite of successful therapy. Symptoms and physical signs of primary disease often overlap with those caused by host's immune response. In such a situation, it is difficult to decide whether therapy of primary disease has failed due to drug resistance or whether persistence or deterioration is the result of immune response. Occasionally pathogen can transform into "superantigen" that may lead to "cytokine storm". Resulting immune-mediated complications may endanger life and at best, treated symptomatically. Immune suppressive drugs such as steroids, chemotherapeutic agents, IVIG or specific antibodies may not be able to suppress undesirable immune response. It is not just the immune suppression that is required but ideally immune modulation. Immune modulation refers to enhancing protective responses while avoiding destructive ones. At present, science falls short of anticipating harmful immune responses and lacks specific immune intervention.Laboratory test results are also dependent on host response and hence need cautious interpretation based on clinical profile in consideration with multiple variables. In final analysis, fight between host and pathogen is a complex one and often unpredictable. It is hoped that most children evoke favourable response but pediatrician has to be watchful even in the most benign disease.

Mechanism Of Oral Tolerance Induced By Bioencapsulated Coagulation Factor IX In Hemophilia B Mice

Currently, the most serious complication in hemophilia therapy is the development of neutralizing antibodies (inhibitors) against the therapeutic coagulation factor. Inhibitor formation in treatment of hemophilia B (deficiency in factor IX, FIX) is less frequent than in hemophilia A. However, FIX inhibitors tend to be high-titer, and 25-50% of such patients develop anaphylactic reactions against FIX. Clinical immune tolerance induction (ITI) protocols (to eliminate the inhibitors) are lengthy, expensive, and are often terminated in hemophilia B due to anaphylaxis or nephrotic syndrome. In order to establish a protocol for prevention of FIX inhibitors in high-risk patients (i.e. those with gene deletions or other severe mutations) while avoiding immune suppressive drugs or genetic manipulation, we have developed a source of bioencapsulated human FIX. Oral delivery of plant leaf material, expressing CTB-FIX in chloroplasts, effectively suppresses inhibitor formation and prevents anaphylaxis in FIX replacement therapy in hemophilia B mice of the C3H/HeJ F9-/- strain (PNAS 107:7101, 2010). This is accomplished by delivery of the fusion molecule to the epithelium of Peyer's Patches (PP), which expresses high levels of GM1, the receptor for the CTB transmucosal carrier. Subsequently, FIX antigen is transferred to dendritic cells (DCs) in the PP and also into the circulation. Mice fed twice per week for 2 months fail to form inhibitors during a 2-month treatment phase (1 IU rFIX, IV, once/week), lack IgE formation and fatal anaphylactic reactions (which occur in control mice), but still form non-inhibitory IgG1 at reduced titers. An oral dose of FIX antigen equivalent to ∼1.5 IU rFIX/kg is sufficient to achieve tolerance. To define the underlying mechanism, we analyzed DC and lymphocyte populations in different compartments (n=4-6 for all experimental groups and data sets). Compared to control mice, which received FIX only or were fed with WT plant material and treated with FIX, tolerized mice had a marked increase in the frequencies of CD103+ DC and pDC in mesenteric lymph nodes (MLN) and PP. Both DC subsets are known to play crucial roles in oral tolerance and Treg induction. A minor, non-significant increase pDCs was also seen in the spleen. Interestingly, tolerized mice also showed a significant increase in the frequencies of CD4+ T cells expressing gut homing receptors (CCR9+ and α4β7), including in the spleen and thus indicating interaction between the gut and systemic immune systems. The spleen is a major site for systemic immune surveillance and critical for responses to intravenous FIX. Splenocytes re-stimulated in vitro with hF.IX were anayzed by RT-PCR array. We found that Th1 and Th2 cytokines (IL2, IL-4, IL-13, IFN-γ) were downregulated by oral FIX delivery, whereas immune suppressive cytokines (IL-10, TGF-β), and the Treg marker Foxp3 were upregulated, suggesting a switch from an effector to a suppressive response. Moreover, the frequency of LAP+CD4+CD25-FoxP3- T cells (which suppress responses via TGF-β) was dramatically increased in FIX-fed mice in PP (from <5% to 10-20%), and to lesser extent in spleen and MLN. Although the overall frequency of CD4+CD25+FoxP3+ Treg was not altered, tolerized mice had a significant increase in LAP+ cells in that population, indicating Treg activation. Interestingly, we failed to prevent anaphylaxis or inhibitor formation in C3H/HeJ [F9-/- x IL-10-/-] mice (n=7 per group, 42% mortality after 4th FIX injection, >5 BU in surviving animals). Therefore, both IL-10 and TGF-βare integral components of the tolerance mechanism. In summary, oral delivery of CTB-FIX antigen bioencapsulated in plant cells prevents anaphylaxis and inhibitor formation against FIX via induction of a complex immune regulatory network, involving increases in Treg-promoting subsets of DC, induction/activation of LAP+ Treg, and active cytokine-mediated suppression. Preliminary data suggest that the regimen can also be used to desensitize mice from FIX. While animal experiments performed thus far have utilized transgenic tobacco cells, we have now generated and characterized FIX-transplastomic lettuce expressing CTB-FIX, which will be tested towards translation of this approach. Disclosures:Daniell:Bayer: Research Funding. Herzog:Genzyme: AAV technology, AAV technology Patents & Royalties.

Covert Warfare Against the Immune System: Decoy Capsids, Stealth Genomes, and Suppressors

In part owing to their low immune profile, adeno-associated virus (AAV) vectors have long been viewed as ideal for in vivo gene transfer. They are based on a small, nonpathogenic virus that is composed of a single-stranded DNA genome and a protein capsid. As compared with many other viral vectors, recombinant AAVs are better at avoiding activation signals, are less efficient in transducing professional antigen-presenting cells, and elicit weaker and more transient innate inflammatory responses.1 Ultimately, these naturally stealthy vectors have been able to express various transgenes for months if not years in numerous immune-competent animal models. Many serotypes with differing tropism that are capable of transducing various cell types in vivo have been identified. Induction of immune tolerance to transgene products has been demonstrated for hepatic gene transfer, which induces regulatory T cells (Tregs).2,3

Early innate immunity determines outcome of Mycobacterium tuberculosis pulmonary infection in rabbits

BackgroundPulmonary infection of humans by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), results in active disease in 5-10% of individuals, while asymptomatic latent Mtb infection (LTBI) is established in the remainder. The host immune responses that determine this differential outcome following Mtb infection are not fully understood. Using a rabbit model of pulmonary TB, we have shown that infection with the Mtb clinical isolate HN878 (a hyper-virulent W-Beijing lineage strain) leads to progressive cavitary disease similar to what is seen in humans with active TB. In contrast, infection with Mtb CDC1551 (a hyper-immunogenic clinical isolate) is efficiently controlled in rabbit lungs, with establishment of LTBI, which can be reactivated upon treatment with immune-suppressive drugs. We hypothesize that the initial interaction of Mtb with the cells of the host response in the lungs determine later outcome of infection.ResultsTo test this hypothesis, we used our rabbit model of pulmonary TB and infected the animals with Mtb HN878 or CDC1551. At 3 hours, with similar lung bacillary loads, HN878 infection caused greater accumulation of mononuclear and polymorphonuclear leukocytes (PMN) in the lungs, compared to animals infected with CDC1551. Using whole-genome microarray gene expression analysis, we delineated the early transcriptional changes in the lungs of HN878- or CDC1551-infected rabbits at this time and compared them to the differential response at 4 weeks of Mtb-infection. Our gene network and pathway analysis showed that the most significantly differentially expressed genes involved in the host response to HN878, compared to CDC1551, at 3 hours of infection, were components of the inflammatory response and STAT1 activation, recruitment and activation of macrophages, PMN, and fMLP (N-formyl-Methionyl-Leucyl-Phenylalanine)-stimulation. At 4 weeks, the CDC1551 bacillary load was significantly lower and the granulomatous response reduced compared to HN878 infection. Moreover, although inflammation was dampened in both Mtb infections at 4 weeks, the majority of the differentially expressed gene networks were similar to those seen at 3 hours.ConclusionsWe propose that differential regulation of the inflammation-associated innate immune response and related gene expression changes seen at 3 hours determine the long term outcome of Mtb infection in rabbit lungs.

Loss of response to biologics versus increased risk of lymphoma in children with inflammatory bowel disease: the clinician’s conundrum

Inflammatory bowel disease in children can be marked by aggressive disease both at presentation and over time. Risk stratification of individual patients may help identify when early biologic therapy is justified. Currently, combination biologic and immunomodulator therapy for moderate-to-severe Crohn’s disease is the most effective treatment regimen. The clinician’s conundrum arises from the recent understanding that rare but serious adverse events do occur with use of these strong immune suppressive drugs and may be more prevalent with combination therapy. An understanding of the natural history of Crohn’s disease and ulcerative colitis and the benefits and risks of the current medical armamentarium is essential to provide optimal care for each child with inflammatory bowel disease.

Homeostatic T Cell Proliferation after Islet Transplantation

Pancreatic islet transplantation in patients with type 1 diabetes mellitus is performed under immunosuppression to avoid alloreactive T cell responses and to control the reactivation of autoreactive memory T cells. However, lymphopenia associated with immunosuppression and T cell depletion can induce a paradoxical expansion of lymphocyte subsets under the influence of homeostatic proliferation. Homeostatic T cell proliferation is mainly driven by the IL-7/IL-7 receptor axis, a molecular pathway which is not affected by standard immune-suppressive drugs and, consequently, represents a novel potential target for immuno-modulatory strategies. In this review, we will discuss how homeostatic T cell proliferation can support autoimmunity recurrence after islet transplantation and how it can be targeted by new therapeutic approaches.

Genome wide analysis of Cyclophilin gene family from rice and Arabidopsis and its comparison with yeast

Cyclophilin proteins are the members of immunophillin group of proteins, known for their property of binding to the immune-suppressant drug cyclosporin A, hence named as cyclophilins. These proteins are characterized by the presence of peptidyl prolyl isomerase (PPIase) domain which catalyzes the cis-trans isomerisation process of proline residues. In the present study, an in-silico based approach was followed to identify and characterize the cyclophilin family from rice, Arabidopsis and yeast. We were able to identify 28 rice, 35 Arabidopsis and 8 yeast cyclophilin genes from their respective genomes on the basis of their annotation as well as the presence of highly conserved PPIase domain. The evolutionary relationship of the cyclophilin genes from the three genomes was analyzed using the phylogenetic tree. We have also classified the rice cyclophilin genes on the basis of localization of the protein in cell. The structural similarity of the cyclophilins was also analyzed on the basis of their homology model. The expression analysis performed using Genevestigator revealed a very strong stress responsive behavior of the gene family which was more prominent in later stages of stress. The study indicates the importance of the gene family in stress response as well as several developmental stages thus opening up many avenues for future study on the cyclophilin proteins.

Immunosuppressive Drugs up-Regulate the Immunoinhibitory Receptor Osteoactivin On Human Monocyte-Derived Dendritic Cells

AbstractAbstract 3284 Introduction:Dendritic cells (DC) abundantly express the type I transmembrane glycoprotein Osteoactivin (OA) - also known as transmembrane glycoprotein NMB and DC-HIL - compared to low expression levels on monocytes. Antigen-presenting cells interact via OA with the type I transmembrane proteoglycan syndecan-4 (SD-4) on T cells which inhibits T cell activation. We previously reported on increased expression of OA upon exposure of monocyte-derived DC (moDC) to immunosuppressive drugs (e.g., Gutknecht et al ASH annual meeting 2011). Here we extended these analyses and comparatively analyzed the impact of various immunsuppressive drugs (ID) on moDC phenotype and function. Methods:The moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Clinically relevant concentrations of ID were added to the culture medium every second day starting with the first day of culture (cyclosporine A: 1μg/ml, prednisolone: 3.5μg/ml, tacrolimus: 10ng/ml, mycophenolat-mofetil 0.1μM, methotrexat 230ng/ml). Cells were harvested for immunophenotyping by flow cytometry, western-blotting and real-time PCR. Cytokine release by moDC was determined on day 7 by ELISA. Functional properties were determined by mixed lymphocyte reactions (MLR) on day 7 of culture. Results:Exposure of moDC to therapeutic concentrations of prednisolone resulted in significantly reduced expression of the costimulatory molecules CD83 and CD86 and increased levels of the monocyte marker CD14, indicative of impaired differentiation. Tacrolimus significantly increased CD14 expression and reduced CD83 expression, while the other ID did not cause significant alterations. All ID altered the release of the immunomodulatory cytokines IL-10, IL-6 and TGF-ß. Notably, all ID except cyclosporine A caused a substantial upregulation of the immunoinhibitory receptor OA in moDC. The extent of OA expression increased over time of exposure to ID during differentiation and resulted in reduced capacity of the moDC to stimulate allogenic T cells which could be restored by disruption of OA/SD-4 interaction using a blocking OA antibody. Conclusion:Increased expression of OA on moDC upon exposure to ID contributes to inhibition of T-cell activation. The mechanisms underlying the differential effect of cyclosporine A are presently under study. Our results indicate that targeting OA/SD-4 interaction may hold promise for modulation of T cell responses in various pathophysiological conditions and immunotherapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Identification of Candidate Transcriptional Biomarkers Associated with Chronic Graft-Versus-Host Disease Following Allogeneic Hematopoietic Cell Transplantation

Abstract 4190 Background:Chronic graft-versus-host disease (cGvHD) and its treatment result in significant morbidity and mortality, limiting the clinical benefit of allogeneic hematopoietic cell transplantation (HCT). The diagnosis of cGvHD can be challenging especially when diagnostic features are absent or when the clinical features are confined to internal organs (i.e., lungs). A noninvasive, diagnostic and prognostic test is needed to determine which pts have or will develop cGvHD. Objective:To identify candidate blood biomarkers using gene expression profiling of the circulating donor immune cell repertoire following allogeneic HCT and develop a classification rule to distinguish cGvHD from non-GvHD pts. Methods:In this cross sectional discovery phase study we identified 63 pts (median age 50 yrs; range 19–64) who were beyond 100 days from transplantation (median 424 days) with complete (100%) donor CD3+ T cell chimerism. Pts were divided into cGvHD and non-cGvHD groups based on conventional criteria. To adjust for the influence of immune suppression (IS) medication a prospective cohort of 7 new onset cGvHD pts who were free of IS drugs at the time of onset of cGvHD and at the time of blood sample acquisition. Peripheral blood mononuclear cells were obtained from the 70 pts on IRB approval protocols. The RNA was processed on the Agilent whole human genome 44k microarray platform. Bioinformatics programs including AILUN, GeneSpring, SAM, and PAM. Results:Three data sets were constructed to address the comparison of cGvHD pts on IS medication to drug free non-GvHD pts. First a ‘screening set', compared gene expression levels in pts with cGvHD on IS (n=14) to those with cGvHD not yet on IS (n=7) using a two-sample t-test and eliminated more than 27k genes whose t-test statistics were beyond the interval of [-1,1] as differences in expression were possibly related to the presence or absence of these medication. We next applied the Prediction Analysis of Microarray (PAM) method to a ‘training set' to discriminate 21 GvHD pts from 21 non-GvHD using the 16,103 selected probes from the screening set and found a minimum misclassification error of 6 samples was achieved with a probe set that contained 10 genes. The candidate genes represented a compensatory response to cGvHD rather than a pro-inflammatory gene signature. We next evaluated the performance of the classification rule from the ‘training set' to differentiate cGvHD pts from non-GvHD pts in the 14 pt ‘test set' and correctly predicted 3 new cGvHD and all 7 non-GvHD controls. We repeated PAM and adjusted for possible confounding variables, that included length in days of sample acquisition date relative to the transplant date, recipient age, donor gender, disease histology (myeloid or lymphoid malignancy), related or unrelated donor to recipient status, and type of conditioning regimen (full dose or reduced intensity conditioning) and observed a result similar to that from the unadjusted analysis. Specifically, adjusted PAM achieved a minimum cross-validated misclassification error rate of 9 among the entire cohort of 70 samples. Finally, the entire analysis was bootstrapped and cross-validated with similar results obtained; the genes with the top frequencies for discriminating cGvHD were over-expression of IL1R2, ADAMTS2 and TS3, TPST1, SESN1, AREG, IRS2, GPER, BCAT1 and CxCR7. IL1R2 is the decoy receptor for the pro-inflammatory IL1 cytokine, ADAMTS2 and TS3 are pro-collagen modifying enzymes that regulate the clipping of collagen molecules to impart structure to connective tissue, AREG is a key gene that underlies the development of oral lichen planus, and pathway analysis revealed that the combination of the other over-expressed genes converge through IL-4, Il-6 and IL-10 signaling pathways which are considered GvHD blocking signals and controllers of inflammation. Conclusion:In the current discovery phase study we used expression profiling of peripheral blood mononuclear cells combined with biostatical analyses and identified several candidate biomarkers that associated with chronic GvHD. The genes and signaling pathways highlighted in the current analysis suggest that compensatory responses that control inflammation or are involved with profibrotic matrix remodeling represent a dominant GvHD “gene footprint”. The identified genes have biological relevance to chronic GvHD and may further the understanding of its pathogenesis. Disclosures:No relevant conflicts of interest to declare.

Suppression of Inhibitor Formation in Protein and Gene Therapy for Hemophilia Using Ex Vivo Expanded Treg

Abstract 13Hemophilia A and B result from deficiency in clotting factor VIII (FVIII) or IX (FIX), respectively. In a subset of patients, treatment by factor replacement therapy is limited by formation of inhibitory antibodies to the clotting factors, representing a serious complication that increases risks of morbidity and mortality. Immune responses to the therapeutic coagulation factors are also a concern in newly emerging gene therapies. Regulatory T cells (Treg) offer to be a novel alternative pathway toward immune tolerance. Treg has been identified as a major component of immune tolerance to coagulation factors in pre-clinical studies. Therefore, we hypothesize that ex vivo expanded autologous Treg can suppress inhibitor formation. Our study seeks to test this approach in hemophilic mice. Initially, we optimized in vitro expansion of murine BALB/c-derived Treg. Using flow sorting, GFP+ cells were purified (>98% purity) from spleens of BALB/c knock-in mice containing a GFP reporter linked to FoxP3 expression with an IRES sequence. Sorted cells were stimulated in culture using anti-CD28/anti-CD3 beads in the presence of high-level IL-2 (1000 U/ml). IL-2 was replenished every second day in culture. After ∼1 week, cells were freshly stimulated. At the end of 2 weeks, viability, purity, and FoxP3/GFP expression was confirmed. Greater than 30-fold expansion was repeatedly accomplished. Assumming a dose of 1×106 Treg/mouse, expansion is sufficiently robust to treat >30 mice starting with Treg from 2–3 donor mice. Ex vivo expanded Tregs were adoptively transferred to male hemophilia A mice (BALB/c F8e16 −/−), which were then treated with F.VIII (1 IU human B domain-deleted F.VIII, IV, once per week) for two months. Bethesda assays demonstrated that Treg transplant had effectively suppressed inhibitor formation. Inhibitor titers in control mice were 15–20 BU at 1 month and 30–40 BU at two months. In contrast, Treg treated mice (n=5 per group) formed at most low-titer inhibitors (2–3 BU for both time points). By 2 months, peripheral Treg frequencies had returned to near baseline. To further demonstrate presence of a Treg population capable of suppressing antibody formation against F.VIII, a secondary transfer of sorted CD4+CD25+ splenocytes was performed. Recipient hemophilia A mice were immunized against F.VIII in adjuvant. Compared to mice receiving control Treg, there was significant (P<0.05) suppression of inhibitor formation against F.VIII. In other experiments, Treg therapy was also able to significantly reduce inhibitor titers in hemophilia A mice with pre-existing F.VIII inhibitors. We chose hemophilia A mice (n=6) that developed on average 25 BU from F.VIII replacement therapy. Half of these received Treg transplant, and all mice received 8 more weeks of F.VIII treatment. Inhibitor titers in the control group increased to ∼100 BU. Treg therapy substantially reduced this response (to 15–20 BU, P<0.001). In order to evaluate Treg therapy for hemophilia B, BALB/c F9 −/− × FoxP3-GFP mice were generated. Treg were isolated from these mice as described above, expanded in vitro, and transferred to (BALB/c F9 −/−) hemophilia B mice. Two days later, to test their effectiveness in a gene therapy setting, these mice were treated by intramuscular injection of AAV1 vector expressing human F.IX. By 6 weeks after gene transfer, control mice had formed high-titer antibody against hF.IX (>20 mg IgG/ml plasma, ∼ 10 BU). In contrast, anti-hF.IX formation was undetectable in mice that had received Treg prior to vector administration (n=4/group). While perhaps not as potent as antigen-specific Treg, our data demonstrate the ability of highly purified and ex vivo expanded bulk Treg to control inhibitor formation and thus support their utility for tolerance induction in hemophilia. Their effectiveness may involve emergence of a more specific Treg population after repeated in vivo exposure to antigen. In gene therapy, Treg transplant may be a more desirable alternative to use of immune suppressive drugs. Providing additional immune regulation around the time of vector administration, i.e. when activation signals are provided to the immune system, could be sufficient to prevent immune rejection long-term while inducing antigen-experienced Treg for durable tolerance. Disclosures:Herzog:Genzyme Corp.: Royalties, AAV-FIX technology, Royalties, AAV-FIX technology Patents & Royalties.

In Vivo Selection of Mycophenolate Mofetil-Resistant T Cells for Adoptive Immunotherapy.

AbstractAbstract 3008In vivo selection of mycophenolate mofetil-resistant T cells for adoptive immunotherapy. BackgroundFollowing allogeneic solid organ or hematopoietic stem cell transplantation, adoptive transfer of therapeutic T cells may be hindered by the requirement for immune suppressive drugs to prevent rejection or graft-versus-host disease. Mycophenolate mofetil (MMF) is a non-competitive inhibitor of inosine-5’-monophosphate dehydrogenase 2 (IMPDH2), an inducible enzyme that generate guanine nucleotides for DNA and RNA synthesis in T cells. In this study, we have evaluated the potential for gene transfer to T cells of a mutated IMPDH2 that confers >2000-fold resistance to MMF (IMPDH2R; T333I, S351Y). MethodsWild type IMPDH2WT, IMPDH2R and IMPDH2 with a catalytic site mutation (IMPDH2CS; C331A) were cloned into SFG retroviral vectors as fusions to eGFP reporter sequences. Murine thymoma (BW 5147) or CD8 T cells were transduced with each vector and their phenotype and function evaluated in the presence or absence of mycophenolic acid (MPA), the active metabolite of MMF. ResultsBW thymoma cells transduced with IMPDH2R exhibited less apoptosis than cells transduced with IMPDH2CS in response to MPA (ratio % Annexin V+ MPA: no MPA- IMPDH2R = 1.2; IMPDH2CS = 2.9, p=0.01). Cells transduced with IMPDH2R were also able to overcome the G1 cell-cycle arrest induced by MPA when compared to control IMPDH2CS cells (Ratio %cells in S-G2/M phases MPA: no MPA- IMPDH2R = 1.0; IMPDH2CS = 0.3, p=0.03). This led to selective enrichment of IMPDH2R transduced cells in the presence of MPA. At low dose MPA (450nM), IMPDH2R transduced cells enriched compared to IMPDH2CS but not IMPDH2WT (ratio % GFP MPA: no MPA- IMPDH2R = 1.6 vs IMPDH2CS = 1.1, p=0.04 and IMPDH2WT = 1.5 p= 0.14). However, at high dose (4500nM) IMPDH2R exhibited enhanced enrichment (ratio % GFP MPA: no MPA- IMPDH2R = 2.8 vs IMPDH2CS = 1.0 p=0.03 and vs IMPDH2WT= 1.5 p=0.03).Gene transfer of IMPDH2R into murine CD8 T cells also led to selective enrichment compared to controls in the presence of MPA when cultured with proliferation-inducing common gamma-chain cytokines (ratio MPA: no MPA IMPDH2R = 4.4 vs. IMPDH2CS = 1.10, p=0.02). Strong selection for IMPDH2R-tranduced CD8 OT-1 T cells in the presence of MPA was also observed under conditions of antigen-induced proliferation (ratio IMPDH2R= 7.1, control = 0.8, p=0.002).To assess in vivo selection, sub-lethally irradiated (2Gy) B6.PL (Thy1.1) mice were injected with a 1:1 mix of OT1 TCR transgenic Thy1.2 CD8 T cells transduced with IMPDH2R or IMPDH2CS that could be differentiated by the congenic markers CD45.1 and CD45.2. Transferred cells were stimulated by s.c. injection with cognate peptide (SIINFEKL) in IFA and MMF (200mg/kg/day) was given by daily ip injection. As in vitro, IMPDH2R-transduced OT1 cells were preferentially selected over IMPDH2CS-transduced cells following MMF treatment (day 14 ratio IMPDH2R to IMPDHCSwas 19.3 vs 1.2 in the absence of MMF). ConclusionsT cells transduced with IMPDH2R are resistant to the anti-proliferative and apoptotic effects of MPA in vitro and demonstrate strong selection in vivo compared to controls at therapeutic levels of MMF. These data support the potential of conferring MMF resistance as a strategy to permit the survival of therapeutic T cells in immunosuppressed allograft recipients. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

Functional characterization of calcineurin homologs PsCNA1/PsCNB1 in Puccinia striiformis f. sp. tritici using a host-induced RNAi system.

Calcineurin plays a key role in morphogenesis, pathogenesis and drug resistance in most fungi. However, the function of calcineurin genes in Puccinia striiformis f. sp. tritici (Pst) is unclear. We identified and characterized the calcineurin genes PsCNA1 and PsCNB1 in Pst. Phylogenetic analyses indicate that PsCNA1 and PsCNB1 form a calcium/calmodulin regulated protein phosphatase belonging to the calcineurin heterodimers composed of subunits A and B. Quantitative RT-PCR analyses revealed that both PsCNA1 and PsCNB1 expression reached their maximum in the stage of haustorium formation, which is one day after inoculation. Using barely stripe mosaic virus (BSMV) as a transient expression vector in wheat, the expression of PsCNA1 and PsCNB1 in Pst was suppressed, leading to slower extension of fungal hyphae and reduced production of urediospores. The immune-suppressive drugs cyclosporin A and FK506 markedly reduced the germination rates of urediospores, and when germination did occur, more than two germtubes were produced. These results suggest that the calcineurin signaling pathway participates in stripe rust morphogenetic differentiation, especially the formation of haustoria during the early stage of infection and during the production of urediospores. Therefore PsCNA1 and PsCNB1 can be considered important pathogenicity genes involved in the wheat-Pst interaction.