Monoclonal OKT3 Research Articles

Exploring BiTE-Integrated CAR T-Cell Therapy to Overcome Tumor Antigen Escape and Reinforce CAR-T Therapy in Mantle Cell Lymphoma

Background T cell engagers (BiTEs) are bispecific T-cell engaging antibodies that redirect T cells to target antigen-expressing tumor cells. Combining a second antigen-targeted bispecific T cell engager (BiTE) with CAR T cells or incorporating bispecific engaging antibody-secreting T cells (engager-T cells) is expected to augment T-cell activation and proliferative capacity, and antitumor cytotoxicity, even towards tumor cells expressing tumor antigens heterogeneously. Here, we report a BiTE-integrated T-cell therapeutic strategy to overcome tumor antigen escape and reinforce the efficacy of CAR-T therapy in mantle cell lymphoma (MCL). Methods CD20-directed bispecific T-cell engager (CD20-BiTE) was constructed with a publicly available VHH fragment from an anti-CD20 single-domain antibody, and anti-CD3 scFv from OKT3 monoclonal antibody. CD19-directed CAR construct is comprised of anti-CD19 scFv from publicly available FMC63 monoclonal antibody, CD8 transmembrane-, 4-1BB costimulatory-, and CD3ζ signaling-domains. Human primary pan-T cells were isolated from PBMC derived from normal human donors (MD Anderson Blood Bank) or patients with MCL. Activated T cells were used for effector functional assay or transduced with lentivirus to produce CAR T or engager-T cells. Effector function of bispecific antibodies and T cells was determined via T-cell activation, cytokine release, and T-cell-dependent cytotoxicity assay in a co-culture model. Results Treatment with CD20-BiTE enhances the effector function of naïve T cells: the BiTE-Fc fusion protein was ectopically expressed in Expi293 and purified by immobilized Protein-A. The binding specificity of scFv(s) of CD20-BiTE was confirmed to bind strongly and specifically to CD20-expressing cells. In vitro functional analyses demonstrated that co-culture of naïve T cells with CD20-exressing cells in the presence of CD20-BiTE induced antigen-specific activation of the T cells (increase in the expression of CD25 and CD69). In the presence of naïve T cells and antigen-positive MCL cells, CD20-BiTE enhanced the secretion of cytokines (IFNγ and TNFα, > 2-fold increase) and CD8 T-cell cytotoxic molecule granzyme B by T cells, in a BiTE-dependent and CD20-specific manner. The enhanced effector function translated into improved antitumor cytotoxicity, as activated T cells treated with CD20-BiTE exerted 43- 72% increase in the killing efficacy of JeKo-1 BTK-KD cells. CD20-BiTE-combined CAR T cells endowed with enhanced antitumor activity overcome tumor antigen escape and resistance to current CD19-CAR T cell therapy: when primary T cells and CD19-CAR T cells were co-cultured with MCL cells, addition of CD20-BiTE potentiated T cell-dependent cytotoxicity ( p < 0.001). Since both CD19-dependent and -independent CAR T cell-resistance often develop during adoptive cell therapy in MCL, we sought to overcome the resistance in MCL by combining a second antigen-targeted CD20-BiTE with CAR T cells or incorporating CD20-BiTE secreting T cells (engager-T cells). Remarkably, when combined with CD19-CAR T cells, CD20-BiTE mediates significant increase in the antitumor cytotoxicity, compared to T cells alone ( p ≤ 0,001 and p < 0.001 for 2nM and 5nM of the BiTE, respectively), in a CD20-specific fashion, in both MCL cells with diminished CD19 expression, and CD19-CAR T-resistant primary MCL cells. Importantly, in a transwell assay, CD19-CAR T cells demonstrated restored potent killing (>3-fold increase over CAR T cells alone) of the CD19-low or CAR T therapy resistant tumor cells, but only when the engager-T cells were present in the insert, suggesting the engaging molecules were able to redirect CD19-CAR T cells to CD20-positive tumor cells regardless of their CD19 expression. The anti-lymphoma efficacy of engager-CAR T cell therapy will be further evaluated in our well-established unique PDX model for MCL. In summary, we have successfully generated a CD20-directed bispecific T-cellengaging antibody and CD20-BiTE engager-T cells that could be combined with CAR T cell therapy, which was demonstrated effective in overcoming tumor antigen escape and resistance to current CD19-CAR T cell therapy in MCL.

Post-Transplant Lymphoproliferative Disorder after Heart Transplant in the Pediatric Population: A Systematic Review

Background: Post-transplant lymphoproliferative disorder (PTLD) is a B-cell proliferation disorder that results from disruption in the physiological mechanisms for proliferation in an immunocompromised host after a solid organ transplant. Our study aims to review the demographic characteristics and clinical outcomes after transplantation. We also aim to study the role of immunosuppression induction therapy, the effect of PTLD on survival, and the effective chemotherapy used for B-cell disorders leading to improved survival. Methods: Following the PRISMA guideline, we performed a comprehensive literature search on PubMed, Cochrane Library, Embase, and clinicaltrials.gov from the past decade on May 04, 2020. We used the MeSH terms of organ transplantation and lymphoproliferative disorders. 1741 articles were screened. We excluded all case reports, case series, pre-clinical trials, review articles, and meta-analysis and included three cohort studies and one prospective multicentric study. We extracted the data for baseline characteristics, the reason for transplantation, recipient & donor EBV status, immunosuppression used, type & stage of PTLD, organ system involved, duration between transplant and PTLD diagnosis, treatment, response to therapy, adverse effects of therapy and mortality. Results: We studied 9617 patients in the included four studies, out of which 499 patients developed PTLD. Data in these studies was collected over the last 20-26 years. Median follow-up of patients since transplant was 3-9 years (average 7.5y). Table 1 The age of the patients ranged from 3-18 years with a male: female gender ratio of 48:52% and around 50% of the patients were seronegative to EBV pretransplant. The following drugs were used for immunosuppression: cyclosporin, tacrolimus, azathioprine, mycophenolate, interleukin 2 receptor antagonist (basiliximab), corticosteroids, anti-thymocyte globulins(ATG). Kindel et al. narrated that the development of eosinophilic oesophagitis may be a marker for the development of PTLD.Gajarski et al. concluded that Post-transplant Immunosuppressive induction therapy with cytolytic drugs (e.g OKT3 monoclonal antibody, ATG, thymoglobulin Basiliximab and daclizumab) , lowers the rate of PTLD, graft rejections, and early infections in post-transplant patients as compared patients who did not receive induction therapy . This depends upon the type of induction e.g OKT3 monoclonal antibody was associated with increased PTLDs and graft rejection, while Thymoglobulin/IL-2R antagonists demonstrated to decrease both the outcomes. Claire et al. described that overall mortality is decreasing due to the ongoing better understanding of pathophysiology and treatment options related to solid organ transplant. The mortality of the post-transplant congenital heart disease group was higher as compared to the cardiomyopathy group due to high comorbidities and surgical complications. The study by Christopher et al. showed that EBV seronegativity before transplant is associated with an increased risk of PTLD. PTLD is associated with lower survival rates as compared to non-PTLD groups. Conclusion: Our review illustrates that pretransplant seronegativity, OKT3 monoclonal antibody, and the development of eosinophilic esophagitis during the immunosuppressive regime increase the risk of PTLD. This study demonstrates that with a better understanding of PTLD and tumor behavior, the all-cause mortality rates are falling significantly. PTLD is one of the leading causes of mortality in post-transplant patients. However, the immunosuppressive induction therapy, absence of eosinophilic esophagitis, thymoglobulin/IL-2R antagonists improve survival and outcomes in the post-transplant patient in terms of graft rejection, graft failure, and development of PTLD. Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.: Honoraria, Research Funding, Speakers Bureau.

Research Highlights

Research Highlights

Abstract 5098: NG-348: a novel oncolytic virus designed to mediate anti-tumour activity via the potent and selective polyclonal activation of tumor-infiltrating T-cells

Abstract NG-348 is a transgene-modified variant of enadenotucirev, a chimeric oncolytic group B adenovirus with potent and selective anti-tumor activity against a range of epithelial cancer cells. Enadenotucirev has a blood stability profile that enables systemic dosing and has been administered intravenously to over 90 cancer patients. These studies have demonstrated that IV dosed enadenotucirev is delivered to tumors and subsequent virus activity is associated with CD8+ T-cell infiltration in tumor cell nests, consistent with immune stimulation within the tumor. NG-348 encodes two immunomodulatory proteins in its genome: full-length human CD80, and a membrane anchored single chain variable fragment of the mouse anti-human CD3ϵ monoclonal antibody OKT3. Together these membrane proteins provide both T-cell receptor (signal 1) and costimulatory (signal 2) activation signals required to polyclonally activate tumor-infiltrating T-cells. When expressed on the surface of NG-348 infected tumor cells the transgenes therefore enhance the potency of the virus by driving local T-cell immune responses selectively in the tumor microenvironment. The expression of both transgenes encoded in the NG-348 virus is controlled by the endogenous virus major late promoter. This restricts expression of the proteins to the surface of cells permissive to virus infection (i.e. tumor cells) and prevents off-target expression in the cells from healthy tissues. Using co-cultures of human T-cells with human tumor cell lines, we have shown that NG-348 infected tumor cells potently activate both CD4 and CD8 T-cells. This was demonstrated by analysis of activation marker expression (CD25, CD69), intracellular and secreted cytokines (IL-2, TNF, IFNγ) and induction of T-cell mediated tumor cell death by apoptosis (prior to oncolytic death by the virus). Treatment of different human non-tumor cells (e.g. fibroblasts, T-cells, PBMCs) with NG-348 did not lead to transgene protein expression or activation of T-cells in co-cultures. NG-348 has also been shown to activate human T-cells in vivo, using a human tumor xenograft model system in immunodeficient mice reconstituted with human PBMCs. Collectively these data indicate that following delivery to tumor tissues of patients, NG-348 oncolytic virus can selectively replicate and express it’s payload of T-cell activating ligands. NG-348 should therefore stimulate potent antigen-independent, polyclonal activation of T-cells already present in the tumor, as well as those recruited into the tumor in response to the virus infection, to drive effective anti-tumor immunity. NG-348 is currently in preclinical development with a first phase I study planned to initiate in Q4 2017. Citation Format: Brian R. Champion, Matthieu Besneux, Nalini Marino, Darren Plumb, Prithvi Kodialbail, Sam Illingworth, Rochelle Lear, Alice C. Brown. NG-348: a novel oncolytic virus designed to mediate anti-tumour activity via the potent and selective polyclonal activation of tumor-infiltrating T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2017-5098

MicroRNA expression profiles in human CD3+ T cells following stimulation with anti-human CD3 antibodies

BackgroundAnti-CD3 therapy can induce immunosuppression by several non mutually exclusive mechanisms that have been proposed to explain the therapeutic effect the administration anti-CD3 mAb, but its immunoregulatory mechanism is still not completely clear. In T cells, microRNAs (miRNAs) regulate several pathways, including those associated with immune tolerance. Here, we report changes in miRNA expression in T cells following treatment with anti-human CD3 antibodies. Peripheral blood mononuclear cells were cultured in the presence of the monoclonal antibody OKT3 or a recombinant fragment of humanized anti-CD3. Following these treatments, the expression profiles of 31 miRNA species were assessed in T cells using TaqMan arrays.ResultsEight of the tested miRNAs (miR-155, miR-21, miR-146a, miR-210, miR-17, miR-590-5p, miR-106b and miR-301a) were statistically significantly up- or down-regulated relative to untreated cells.ConclusionsStimulation of T cells with anti-human CD3 antibodies alters miRNA expression patterns, including of miRNA species associated with immune regulatory pathways.

English

Heart transplantation has emerged as the definitive therapy for patients with end-stage cardiomyopathy. The two most common forms of cardiac disease that lead to transplantation are ischemic cardiomyopathy and dilated cardiomyopathy, which together comprise approximately 90% of cases. The other less common forms of heart disease include viral cardiomyopathy, infiltrative cardiomyopathy, postpartum cardiomyopathy, valvular heart disease and congenital heart disease. KEYWORDS: Anesthetic management; Cardiomyopathy; Cardiopulmonary bypass; Heart failure; Heart transplantation; Pulmonary artery hypertension; INTRODUCTION: Heart transplantation has emerged as the definitive therapy for patients with end- stage cardiomyopathy. The two most common forms of cardiac disease that lead to transplantation are ischemic cardiomyopathy and dilated cardiomyopathy, which together comprise approximately 90% of cases. The other less common forms of heart disease include viral cardiomyopathy, infiltrative cardiomyopathy, postpartum cardiomyopathy, valvular heart disease and congenital heart disease. The introduction of cyclosporine in 1981 and monoclonal antibody okT3 in 1983 for heart transplant patients greatly accelerated the progress of organ transplantation.1 The peak period of heart transplantation was mid-1980s.The pace slowed down due to the limitations in the availability of donor organs. We have performed five heart transplant surgeries at Rajiv Gandhi Government General Hospital, Chennai, India during 2009 to 2011. The corresponding author was a member of the team during three cardiac transplant surgeries while the co-author was a member during all the five transplant surgeries. This article is intended to share our experiences and anesthetic challenges during cardiac transplantations.

Liver Allograft Provides Immunoprotection for the Cardiac Allograft in Combined Heart-Liver Transplantation.

When transplanted simultaneously, the liver allograft has been thought to have an immunoprotective role on other organs; however, detailed analyses in simultaneous heart-liver transplantation (SHLT) have not been done to date. We analyzed patient outcomes and incidence of immune-mediated injury in 22 consecutive SHLT versus 223 isolated heart transplantation (IHT) recipients between January 2004 and December 2013, by reviewing 3912 protocol- and indication-specific cardiac allograft biopsy specimens. Overall survival was similar (86.4%, 86.4%, and 69.1% for SHLT and 93.3%, 84.7%, and 70.0% for IHT at 1, 5, and 10 years; p = 0.83). Despite similar immunosuppression, the incidence of T cell-mediated rejection (TCMR) was lower in SHLT (31.8%) than in IHT (84.8%) (p < 0.0001). Although more SHLT patients had preexisting donor-specific HLA antibody (22.7% versus 8.1%; p = 0.04), the incidence of antibody-mediated rejection was not different in SHLT compared with IHT (4.5% versus 14.8%, p = 0.33). While the left ventricular ejection fraction was comparable in both groups at 5 years, the incidence and severity of cardiac allograft vasculopathy were reduced in the SHLT recipients (42.9% versus 66.8%, p = 0.03). Simultaneously transplanted liver allograft was associated with reduced risk of TCMR (odds ratio [OR] 0.003, 95% confidence interval [CI] 0-0.02; p < 0.0001), antibody-mediated rejection (OR 0.04, 95% CI 0-0.46; p = 0.004), and cardiac allograft vasculopathy (OR 0.26, 95% CI 0.07-0.84; p = 0.02), after adjusting for other risk factors. These data suggest that the incidence of alloimmune injury in the heart allograft is reduced in SHLT recipients.

Impact of lithium alone and in combination with antidepressants on cytokine production in vitro.

Lithium is an important psychopharmacological agent for the treatment of unipolar as well as bipolar affective disorders. Lithium has a number of side effects such as hypothyroidism and aggravation of psoriasis. On the other hand, lithium has pro-inflammatory effects, which appear beneficial in some disorders associated with immunological deficits, such as human immunodeficiency virus (HIV) infection and systemic lupus erythematosus (SLE). Therefore, immunological characteristics of lithium may be an important consideration in individualized therapeutic decisions. We measured the levels of the cytokines interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-22, IL-17 and tumour necrosis factor (TNF)-α in the stimulated blood of thirty healthy subjects supplemented with lithium alone, the antidepressants citalopram, escitalopram or mirtazapine alone, the combination of each antidepressant with lithium, and a no drug control. These drugs were tested under three blood stimulant conditions: murine anti-human CD3 monoclonal antibody OKT3 and the 5C3 monoclonal antibody (OKT3/5C3), phytohemagglutinin (PHA), and unstimulated blood. Lithium, alone and in combination with any of the tested antidepressants, led to a consistent increase of IL-1ß, IL-6 and TNF-α levels in the unstimulated as well as the stimulated blood. In the OKT3/5C3- and PHA-stimulated blood, IL-17 production was significantly enhanced by lithium. Lithium additionally increased IL-2 concentrations significantly in PHA-stimulated blood. The data support the view that lithium has pro-inflammatory properties. These immunological characteristics may contribute to side effects of lithium, but may also explain its beneficial effects in patients suffering from HIV infection or SLE.

Modulation of Cytokine Production by Drugs with Antiepileptic or Mood Stabilizer Properties in Anti-CD3- and Anti-CD40-Stimulated BloodIn Vitro

Increased cytokine production possibly due to oxidative stress has repeatedly been shown to play a pivotal role in the pathophysiology of epilepsy and bipolar disorder. Recent in vitro and animal studies of valproic acid (VPA) report antioxidative and anti-inflammatory properties, and suppression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α. We tested the effect of drugs with antiepileptic or mood stabilizer properties, namely, primidone (PRM), carbamazepine (CBZ), levetiracetam (LEV), lamotrigine (LTG), VPA, oxcarbazepine (OXC), topiramate (TPM), phenobarbital (PB), and lithium on the production of the following cytokines in vitro: interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-17, IL-22, and TNF-α. We performed a whole blood assay with stimulated blood of 14 healthy female subjects. Anti-human CD3 monoclonal antibody OKT3, combined with 5C3 antibody against CD40, was used as stimulant. We found a significant reduction of IL-1 and IL-2 levels with all tested drugs other than lithium in the CD3/5C3-stimulated blood; VPA led to a decrease in IL-1β, IL-2, IL-4, IL-6, IL-17, and TNF-α production, which substantiates and adds knowledge to current hypotheses on VPA's anti-inflammatory properties.

Impact of Antidepressants on Cytokine Production of Depressed Patients in Vitro

The interplay between immune and nervous systems plays a pivotal role in the pathophysiology of depression. In depressive episodes, patients show increased production of pro-inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. There is limited information on the effect of antidepressant drugs on cytokines, most studies report on a limited sample of cytokines and none have reported effects on IL-22. We systematically investigated the effect of three antidepressant drugs, citalopram, escitalopram and mirtazapine, on secretion of cytokines IL-1β, IL-2, IL-4, IL-6, IL-17, IL-22 and TNF-α in a whole blood assay in vitro, using murine anti-human CD3 monoclonal antibody OKT3, and 5C3 monoclonal antibody against CD40, to stimulate T and B cells respectively. Citalopram increased production of IL-1β, IL-6, TNF-α and IL-22. Mirtazapine increased IL-1β, TNF-α and IL-22. Escitalopram decreased IL-17 levels. The influence of antidepressants on IL-2 and IL-4 levels was not significant for all three drugs. Compared to escitalopram, citalopram led to higher levels of IL-1β, IL-6, IL-17 and IL-22; and mirtazapine to higher levels of IL-1β, IL-17, IL-22 and TNF-α. Mirtazapine and citalopram increased IL-22 production. The differing profile of cytokine production may relate to differences in therapeutic effects, risk of relapse and side effects.

Long-term stability of T-cell activation and transduction components critical to the processing of clinical batches of gene-engineered T cells

The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan). Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time. We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years. High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies.

Soluble CD200 Is Critical to Engraft Chronic Lymphocytic Leukemia Cells in Immunocompromised Mice

CD200 is a transmembrane molecule with an important immunoregulatory role that is overexpressed on most chronic lymphocytic leukemia (CLL) cells. In this study, we characterized a previously unknown soluble form of this molecule in human plasma termed sCD200. Levels of sCD200 were elevated in the plasma of patients with CLL as compared with healthy controls, and there was a significant correlation with CLL disease stage. Infusion of sCD200(hi) CLL plasma into severely immunocompromised NOD.SCIDγ(c)(null) (NSG) mice enhanced the engraftment of CLL splenocytes as compared with mice receiving sCD200(lo) normal plasma. CLL cells were detected in both the spleen and peritoneal cavity of animals for up to 75 days. Engraftment of CLL cells did not occur after infusion of CLL plasma depleted of sCD200 and was abolished in mice treated with anti-CD200 or OKT3 monoclonal antibody (mAb), suggesting a role for both sCD200 and T cells in CLL engraftment. Notably, anti-CD200 mAb was as effective as rituximab in eliminating engrafted CLL cells when administered 21 days after engraftment. Taken together, our findings point to sCD200 as a novel prognostic marker and therapeutic target for CLL. Furthermore, the humanized mouse model described here may prove valuable to preclinically assess new treatment regimens for CLL.

Ex Vivo Expansion of Human CD8+ T Cells Using Autologous CD4+ T Cell Help

BackgroundUsing in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model.Methods/Principal FindingsWe developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells.ConclusionsWe have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro.

Long-Term Survival of Salvage Cardiac Transplantation for Infective Endocarditis

Cardiac transplantation has been rarely performed in patients with infective endocarditis. A 31-year-old man developed aortic endocarditis due to Brucella melitensis. He presented with fever and developed acute myocardial infarct, severe aortic regurgitation, and heart failure. Aortic valve replacement did not improve cardiac function; hence, an emergent cardiac transplantation was carried out. Eighteen years later, he is doing well and living an active and productive life. Only 6 patients have received a cardiac transplant as part of the treatment of active infective endocarditis. This patient shows how cardiac transplantation may be successfully used as salvage therapy for patients with infective endocarditis who are not candidates for valve replacement or have severe and irreversible myocardial damage.

Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [(3) H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4(+) lymphocyte proliferation but did not affect the proliferation of CD8(+) cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response.

The Registry of the International Society for Heart and Lung Transplantation: Twenty-seventh official adult heart transplant report—2010

The Registry of the International Society for Heart and Lung Transplantation: Twenty-seventh official adult heart transplant report—2010

Heterogeneity of induction therapy in Spain: changing patterns according to year, centre, indications and results

Background. The use of induction drugs has increased markedly over the last 15 years in the USA, but there are few data about their use in other countries. Moreover, there are not enough data about when they are indicated and their long-term effects. The aim of our study was to know the rates of use and the drugs used as induction therapy, in which patients they were prescribed and the long-term graft survival effect in Spain.Methods. We conducted a retrospective cohort study with adult patients (4861) receiving a kidney allograft in Spain over four different years (1990, 1994, 1998 and 2002) with a functioning graft at the end of the first post-transplant year. Induction therapy was defined as when the patient received polyclonal antibodies, OKT3 monoclonal antibodies or anti-CD25 monoclonal antibodies.Results. From 1990 to 2002, the use of induction therapy in Spain changed, with a progressive reduction in the use of OKT3 and an increasing use of anti-CD25 antibodies. There were great differences in the rate of induction use from one centre to another, although with a common trend to greater use at each centre. Induction therapy was mainly prescribed in patients with a higher rejection risk (higher panel reactive antibody (PRA) titres and mismatches and re-transplants) and in older and diabetic recipients. Lastly, patients who were treated with induction therapy had significant higher allograft survival than those who did not (P value = 0.035).Conclusions. The use of induction therapy in Spain has changed, with an increasing use of monoclonal antibodies in recent years. Induction therapy has a protective role in long-term graft survival.

Suppressive activity of epinastine hydrochloride on TARC production from human peripheral blood CD4+ T cells in-vitro

Thymus- and activation-regulated chemokine (TARC) is an important molecule in the development and maintenance of allergic diseases. However, there is little information about the influence of anti-allergic agents on TARC production. The aim of this study is to examine the influence of epinastine hydrochloride, an H1-receptor antagonist, on TARC production from human peripheral blood CD4+ T cells using an in-vitro cell culture technique. CD4+ T cells prepared from healthy subjects were cultured in wells coated with a combination of OKT3 and anti-CD28 monoclonal antibody in the presence or absence of epinastine HCl for 24 h. The cells were also stimulated with interleukin (IL)-4 in a similar manner. Levels of TARC and IL-4 in culture supernatants were examined by ELISA. The addition of epinastine HCl exerted a dose-dependent suppressive effect on the production of both TARC and IL-4 from CD4+ T cells under co-stimulatory molecule stimulation. The minimum concentration of the agent showing a significant suppressive effect on TARC and IL-4 production was 5.0 microM and 2.5 microM, respectively. Epinastine HCl also suppressed the ability of cells to produce TARC in response to IL-4 stimulation, when the agent was added to cell cultures at more than 2.5 microM. It was concluded that this inhibitory action of epinastine HCl may be partially responsible for epinastine's attenuating effect on allergic diseases.

Rapid Reconstitution of Human Antibody Secreting Cells After Haploidentical Allogeneic Stem Cell Transplantation.

Abstract 3682Poster Board III-618Innate immunity including granulocytes, monocytes, and NK cells is reported to recover rapidly after allogeneic stem cell transplantation within weeks. In contrast, adaptive immunity, including T- and B-cells, has delayed recovery over months. In murine models innate type marginal zone and B1 B cells, established at fetal age and providing natural antibodies, are distinguished from adaptive B2 or follicular B cells. A crucial maturation and survival factor for adaptive murine B cells was shown to be TNF-family member BAFF (B cell-activating factor), while development of innate B1 B cells is BAFF independent. Kinetics in reconstitution of innate and adaptive immunity after ablation in adults may give insight into hierarchy and attribution to innate and adaptive immunity of defined lymphocyte populations. Reconstitution of lymphopoiesis after CD3 and CD19 depleted haploidentical stem cell transplantation was analyzed in 10 patients, which received monoclonal anti-CD3 antibody OKT3 as immunosuppressant only. This model may enable detailed in vivo evaluation of de novo B cell formation. Weekly samples before and after reduced-intensity conditioning were analyzed by flow cytometry for absolute numbers of T-cell, NK-, and B-cell subsets. Their origin of host or donor hematopoiesis was differentiated by HLA-FACS. Antibody secreting cells (ASC) were enumerated by ELISPOT. Plasma cytokine concentrations were determined by bead based arrays and ELISA. Complete reconstitution of allogeneic NK cells was found at day +21 after transplantation. CD4+ and CD8+ T-cells and their subsets had delayed reconstitution with less then 100 cells/μl at 3 months after transplantation. CD19+ B-lymphocytes of naïve and memory phenotype (>0,5% of all lymphocytes) were detected not before day +60. In contrast, complete reconstitution of antibody-secreting cells after a nadir (<0,05/μl) was observed at day +14. Absolute numbers of ASC were comparable to those of healthy controls (d+14: 72 ASC/μl vs. control: 12 ASC/μl). ASC secreting the isotypes IgM and IgA were more prevalent than IgG compared to controls (time increase: IgM 20; IgA 10; IgG 2,9). These ASC appear CD19low/neg, CD38+, and intracellular Ig+ in flow cytometry and carried donor HLA-haplotype. Reconstitution of ASC occurred without detectable circulating T-cells and before increase of BAFF concentrations were observed. In summary, the rapid and complete reconstitution of peripheral blood ASC after allogeneic transplantation, far proceeding detection of naïve and memory type B-cells, is a novel observation. Incidence before T-cell reconstitution and increase in BAFF concentrations indicates a T-cell and BAFF independent mechanism allocating these early ASC to innate immunity, potentially maintaining natural antibody levels. Disclosures:No relevant conflicts of interest to declare.

Immunotherapy with Anti-CD3 x Anti-CMV Bispecific Antibody (CMVBi) Armed Donor Derived Activated T Cells (ATC) – A Novel Strategy against Cytomegalovirus (CMV) Post Allogeneic Stem Cell Transplantation (SCT).

Abstract 1157Poster Board I-179 IntroductionCMV reactivation and infection can cause profound negative outcome post allogeneic SCT. Current management strategies against CMV are suboptimal and are associated with adverse effects. Induction of anti-CMV T cell responses by vaccination has not been helpful in immunocompromised hosts. Immunotherapy with CMV specific donor-derived cytotoxic T lymphocytes (CTL) is effective after allografting, but it is expensive, labor intensive, and diffcult to replicate in most centers. Non-toxic targeted therapy is needed to improve clinical outcomes. In earlier studies, we generated anti-CD3 chemically heteroconjugated with anti-Her2/neu or anti-CD20 bispecific antibody (BiAb). Ex vivo expanded anti-CD3 activated T cells (ATC) and armed with BiAb exhibit high levels of specific cytotoxicity directed at the respective tumor antigens. We propose a novel strategy of using ATC armed ex-vivo with engineered CMVBi to target CMV antigens. We tested the strategy initially in an in vitro cell culture model using CMV-infected fibroblasts and T cells from normal human donors. We hypothesize that donor anti-CD3 ATC armed with CMVBi will target and eliminate CMV-infected target cells. Materials and methodsNormal donor peripheral blood mononuclear cells (PBMC) were used to generate ATC by activation with anti-CD3 (OKT3) and interleukin 2 (IL-2). CMVBi was created by chemical heteroconjugation of OKT3 (murine IgG2a) monoclonal antibody and polyclonal anti-CMV (Cytogam®). Specific cytotoxicity was tested by 51Cr release assay using CMV-infected or uninfected human fibroblasts as target cells. Effector populations tested included CMVBi armed ATC and ATC alone; additional controls included CMVBi alone, Cytogam® alone, CMVBi armed, and unarmed PBMC. Cytotoxicity was assessed for CMVBi and an irrelevant BiAb at arming doses ranging from 1 to 500 ng/106 ATC with effector to target ratios (E:T) ranging from 25:1 to 3.125:1. Interferon gamma (IFNγ) EliSpots were used to determine cytokine response after exposing CMV-infected and uninfected fibroblasts to unarmed and CMVBi-armed ATC. ResultsCMVBi arming with as little as 1 ng/106 ATC was significantly more cytotoxic for target cells than unarmed ATC. There was an incremental increase in cytotoxicity with CMVBi armed ATC as the mutiplicity of infection (MOI) was increased in target cells. At all E:T ratios (25:1, 12.5:1, 6.25:1, 3.125:1 ), ATC armed with a dose of 50 ng/106 CMVBi demonstrated markedly enhanced killing of CMV-infected targets (MOI 1) compared to unarmed ATC (see table). In the uninfected control cells, both unarmed and armed ATC lysis was barely detectable over spontaneous lysis. Immunofluorescent studies showed that CMVBi-armed ATC specifically aggregated around GFP fluorescence-marked CMV-infected fibroblasts, whereas unarmed ATC did not aggregate. Cytokine secretion analyzed by IFNγ EliSpot confirmed the superior cytotoxicity of CMVBi-armed ATC. ConclusionWe used polyclonal Cytogam® to make CMVBi-armed ATC that were able to kill target cells expressing various CMV antigens with high specificity. CMVBi-armed PBMC was only minimally cytotoxic in comparision to CMVBi-armed ATC. Hence, infusion of CMVBi alone to patients is unlikely to be as effective as infusion of ATC armed ex-vivo with CMVBi. We demonstrated similar degree of cytotoxicity using different donor ATC including CMV sero-negative donors. This effect is independent of MHC mediated antigen presentation hence, overcomes CMV immune escape. This non-MHC restricted specific killing strategy could be easily adapted for the prevention and/or treatment of CMV infection/disease after allogeneic SCT using donor-derived ATC. Our current use of BiAb-armed ATC for cancer immunotherapy in humans illustrates the feasibility of adoptive immunotherapy with CMVBi-armed ATC in SCT.Mean ± SEM % Cytotoxicity of CMV-infected Targets (ATC from 3 Donors)Effector:Target RatioCMVBi-Armed ATCUnarmed ATCSigned Rank Test (p value)25:189±224±40.00312.5:195±327±4<0.0016.25:186±220±40.0033.125:171±318±20.007 [Display omitted] DisclosuresLum:Transtarget Corporation: Founder.