Anastasia Papadopoulou

Activity of Broad-Spectrum T Cells as Treatment for AdV, EBV, CMV, BKV, and HHV6 Infections after HSCT

Rapidly generated broad-spectrum T cells can simultaneously treat multiple viral infections after hematopoietic stem cell transplant. Killing Multiple Viruses with One Stone Bone marrow or stem cell transplantation is becoming increasingly common for cancer as well as for other blood disorders and genetic diseases. Although patient outcomes are often good and are continuing to improve as technology evolves, the patients are still at risk for a variety of complications. One of the deadliest complications for newly transplanted patients is infection due to their severely compromised immune function. Viral infections are especially problematic, because many viruses have no specific treatments. In a small clinical trial, Papadopoulou et al. demonstrated a way to quickly generate antiviral T cells and give them to transplant patients, to help them safely clear up to four (and potentially five) simultaneous viral infections. It remains difficult to treat the multiplicity of distinct viral infections that afflict immunocompromised patients. Adoptive transfer of virus-specific T cells (VSTs) can be safe and effective, but such cells have been complex to prepare and limited in antiviral range. We now demonstrate the feasibility and clinical utility of rapidly generated single-culture VSTs that recognize 12 immunogenic antigens from five viruses (Epstein-Barr virus, adenovirus, cytomegalovirus, BK virus, and human herpesvirus 6) that frequently cause disease in immunocompromised patients. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term.

Safety and clinical efficacy of rapidly-generated trivirus-directed T cells as treatment for adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant

Adoptive transfer of virus-specific T cells can prevent and treat serious infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv) after allogeneic hematopoietic stem cell transplant. It has, however, proved difficult to make this approach widely available since infectious virus and viral vectors are required for T cell activation, followed by an intensive and prolonged culture period extending over several months. We now show that T cells targeting a range of viral antigens derived from EBV, CMV, and Adv can be reproducibly generated in a single culture over a 2-3-week period, using methods that exclude all viral components and employ a much-simplified culture technology. When administered to recipients of haploidentical (n = 5), matched unrelated (n = 3), mismatched unrelated (n = 1) or matched related (n = 1) transplants with active CMV (n = 3), Adv (n = 1), EBV (n = 2), EBV+Adv (n = 2) or CMV+Adv (n = 2) infections, the cells produced complete virological responses in 80%, including all patients with dual infections. In each case, a decrease in viral load correlated with an increase in the frequency of T cells directed against the infecting virus(es); both immediate and delayed toxicities were absent. This approach should increase both the feasibility and applicability of T cell therapy. The trial was registered at www.clinicaltrials.gov as NCT01070797.

Mesenchymal stem cells are conditionally therapeutic in preclinical models of rheumatoid arthritis

Objective The role of mesenchymal stem cells (MSC) in experimental arthritis is undoubtedly conflicting. This study explored the effect of bone marrow-derived MSC in previously untested and pathogenetically different models of rheumatoid arthritis (RA). Methods MSC were tested both in an induced (adjuvant-induced) and a spontaneous (K/BxN) arthritis model. Arthritis was assessed clinically and histologically. The proliferation of splenocytes and fibroblast-like synoviocytes (FLS) in the presence of MSC was measured by radioactivity incorporation. Toll-like receptor (TLR) expression was measured by real-time PCR. T-regulatory cell (Treg) frequency, T-cell apoptosis and cytokine secretion were monitored by flow cytometry. Results MSC, in vitro, strongly inhibited critical cell populations; splenocytes and FLS. In contrast, MSC proved ineffective in vivo, unless they were administered before disease onset, an effect implying that the inflammatory arthritic milieu potentially abrogates MSC immunomodulatory properties. In order to alleviate inflammation before MSC infusion, the authors administered, at arthritis onset, a short course with a proteasome inhibitor, bortezomib, whereas MSC were infused when established disease was expected. The bortezomib plus MSC group demonstrated a significantly decreased arthritis score over arthritic, MSC-only, bortezomib-only groups, also confirmed by histology and immunohistochemistry. The bortezomib plus MSC combination restored TLR expression and Treg frequency in blood and normalised FLS and splenocyte proliferation, apoptosis and cytokine secretion. Conclusion MSC lose their immunomodulatory properties when infused in the inflammatory micromilieu of autoimmune arthritis. Conditioning of the recipient with bortezomib alters the disease microenvironment enabling MSC to modulate arthritis. Should milieu limitations also operate in human disease, this approach could serve as a strategy to treat RA by MSC.

The proteasome inhibitor bortezomib drastically affects inflammation and bone disease in adjuvant‐induced arthritis in rats

OBJECTIVE To explore the effect of bortezomib in splenocytes and fibroblast-like synoviocytes (FLS) and its in vivo potency in a rat model of adjuvant-induced arthritis (AIA), which resembles human rheumatoid arthritis (RA). METHODS AIA was induced with Freunds complete adjuvant. Splenocyte and FLS proliferation and apoptosis were measured by radioactivity incorporation and flow cytometry, respectively. The invasiveness of FLS from rats with AIA was tested in a Transwell system. The pattern of cytokine secretion was evaluated by cytometric bead array in splenocyte supernatants. Bortezomib was administered prophylactically or therapeutically, and arthritis was assessed clinically and histologically. Immunohistochemistry was performed for markers of inflammation and angiogenesis in joints. Hematologic and biochemical parameters were tested in peripheral blood (PB). Representative animals were examined by computed tomography (CT) scanning before and after bortezomib administration. The expression of Toll-like receptor 2 (TLR-2), TLR-3, and TLR-4 in PB and FLS was measured by real-time polymerase chain reaction, and alterations in specific cell populations in PB and spleen were determined by flow cytometry. RESULTS In vitro, bortezomib exhibited significant inhibitory and proapoptotic activity in splenocytes and FLS from rats with AIA, altered the inflammatory cytokine pattern, and reduced the invasiveness of FLS from rats with AIA. In vivo, bortezomib significantly ameliorated disease severity. Remission was associated with improved histology and decreased expression of CD3, CD79a, CD11b, cyclooxygenase 1, and factor VIII in target tissues as well as down-regulation of TLR expression in PB and cultured FLS. CT scanning demonstrated a bone healing effect after treatment. CONCLUSION Our findings suggest that bortezomib affects AIA in a pleiotropic manner and that this drug may be effective in RA.

Mobilization of Hematopoietic Stem Cells in a Thalassemic Mouse Model: Implications for Human Gene Therapy of Thalassemia

Granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cells may become the preferable source of hematopoietic stem cells (HSCs) for gene therapy because of the higher yield of cells compared with conventional bone marrow harvesting. A G-CSF-associated risk of splenic rupture has been recognized in normal donors of HSCs, but limited information is available about the G-CSF effect in the presence of splenomegaly and extramedullary hematopoiesis. We investigated the G-CSF effect in a thalassemic mouse model (HBB(th-3)) as compared with a normal strain (C57BL/6), in terms of safety, mobilization efficacy, and distribution of stem cells among hematopoietic compartments. There was no death or clinical sequelae of splenic rupture in G-CSF-treated animals of either strain; however, hemorrhagic infarcts in the spleen were detected with low frequency in G-CSF-treated HBB(th-3) mice (12.5%). HBB(th-3) mice mobilized less effectively than C57BL/6 mice (Lin(-)Sca-1(+)c-Kit(+) cells/microl of peripheral blood mononuclear cells [PBMCs]: 90 +/- 55 vs. 255 +/- 174, respectively, p = 0.01; CFU-GM/ml PBMCs: 390 +/- 262 vs. 1131 +/- 875, p = 0.01) because of increased splenic trapping of hematopoietic stem and progenitor cells (Lin(-)Sca-1(+)c-Kit(+) cells per spleen (x10(5)): 487 +/- 35 vs. 109 +/- 19.6, p = 0.01; CFU-GM per spleen (x10(2)): 1470 +/- 347 vs. 530 +/- 425, p = 0.0006). Splenectomy restored the mobilization proficiency of thalassemic mice at comparable levels to normal mice and resulted in the development of a hematopoietic compensatory mechanism in the thalassemic liver that protected splenectomized mice from severe anemia. Our data imply that, in view of human gene therapy for thalassemia, either multiple cycles or alternative ways of mobilization may be required for a sufficient yield of transplantable HSCs. In addition, strategies to minimize the risk of G-CSF-induced splenic infarcts should be explored in a clinical setting.

Adoptive transfer of Aspergillus-specific T cells as a novel anti-fungal therapy for hematopoietic stem cell transplant recipients: Progress and challenges

Although newer antifungal drugs have substantially altered the natural history of invasive aspergillosis, the disease still accounts for significant morbidity and mortality in hematopoietic stem cell transplant recipients. Both the evidence supporting a protective role of T cells against this fungal pathogen and the documented efficacy of adoptive transfer of antigen-specific T cells for prophylaxis and treatment of viral infections post-transplant have stimulated much interest towards development of Aspergillus-specific T cells (Asp-STs) for adoptive immunotherapy in the allogeneic transplant setting. In contrast to the remarkable progress with virus-specific T cells, clinical development of fungus-specific T cells is still in its infancy. Several groups have characterized Asp-STs in healthy individuals and patients with malignant hematological diseases, while others sought to develop GMP-compliant methods of expanding or bioengineering Asp-STs ex vivo as immunotherapy. This review highlights the recent advances in this field, and discusses critical issues involved in development and protocol design of Asp-ST immunotherapy.

T-cell therapy: a powerful tool for the management of viral infections and relapse post hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for many hematological malignancies and genetic disorders. However, infections and relapse of the original disease continue to cause significant morbidity and mortality after transplantation. The observation that HSCT recipients who receive a T-cell replete graft from a human leukocyte antigen (HLA)-matched donor have a lower incidence of relapse and virus-associated disease led to the suggestion that T cells play a protective role in vivo, and formed the basis for subsequent studies to address whether adoptive T-cell transfer could be more broadly applied as a therapeutic tool [1]. This report outlines various T-cell transfer strategies that have been employed clinically and discusses future directions. T cells are one of the main effectors of the immune system and serve in immunocompetent individuals as a defense against viral infections as well as protecting against the development of neoplastic lesions. They are activated via their T-cell receptor (TCR), which binds to antigenic peptides presented in the context of HLA molecules on the surface of target cells. Following the recognition of peptide-loaded HLA class I molecules, activated CD8 cytotoxic T cells can efficiently destroy viral/tumor target cells, while helper CD4 T cells, which are activated upon exposure to peptide-loaded HLA class II molecules, produce effector cytokines that sustain the expansion and effector function of their cytolytic CD8 partners [2]. The first efforts to adoptively transfer effector T cells relied on the premise that peripheral blood isolated from stem cell donors would contain T-cell populations with the requisite anti-tumor and/or antiviral activity. Accordingly, donor lymphocyte infusions have been extensively used in the allogeneic HSCT setting [3]. However, the high ratio of alloreactive to virus/tumor-specific T cells is problematic, especially in recipients of haploidentical transplants in whom a higher incidence of graft-versus-host disease limits the tolerable donor lymphocyte infusion dose, thereby severely restricting the dose of potentially therapeutic T cells [4]. In order to reduce the toxicity and increase the efficacy of adoptive immunotherapy, a number of groups have sought to selectively isolate or amplify specific T-cell therapy: a powerful tool for the management of viral infections and relapse post hematopoietic stem cell transplantation

426. Multi-Pathogen-Specific T Cells: A Single T-Cell Product Simultaneously Targeting Multiple Pathogens for Adoptive Immunotherapy

Viral infections, most commonly by cytomegalovirus (CMV), Epstein-Barr virus (EBV), polyoma virus type I(BK), andfungal infections, mainly by Aspergillus Fumigatus (Asp), are among the deadliest complications for patients undergoing allogeneic hematopoieticstem cell transplantation. Treatment with antiviral and antifungal pharmacological agents, which are todays standard therapy, is often ineffective or toxic whereas it can lead to the outgrowth of drug-resistant strains. Adoptive immunotherapy with the use of antigen-specific T-cells to restore antigen-specific immunity post-transplant, offers an attractive alternative approach to conventional drugs. We here, aimed to generate at large scale, multipathogen-specific T cells (mp-STs) that simultaneously target CMV, EBV, BK and Asp, from healthy donors, with a rapid, simplified, low-cost and minimally laborious protocol. A total of 1,5×107mononuclear cells, derived from 15-20ml blood of eight CMV and EBV seropositive donors, were pulsed with viral pepmixes(CMV: IE1, pp65;EBV: EBNA1, LMP2, BZLF1;BK: Large T, VP1) combined with either Asp lysate or Asp pepmixes (Crf1, Gel1 and SHMT) and cultured in the presence of IL-4/IL-7 for 10 days in G-rex bioreactors. The cells were characterized immunophenotypically by flow cytometry and their specificity/functionality was assessed by IFN-γ Elispot assay. Cells stimulated with either Asp lysate (n=4) or Asp pepmixes (n=4), had a similarexpansion, reaching a mean of 170±36×106and 206±46×106 cells, respectively. All cell lines were polyclonal, comprised predominantly of CD4+ cells (70±5% and 72±3% respectively) and CD8+ cells (19±4% and 18±2% respectively) and expressed central (CD45RA-/CD62L+: 36±6%, and 35±2% respectively) and effector memory markers (CD45RA-/CD62L-: 53±5%, and 54±3% respectively). Importantly, allmp-STs lines (8/8) were specific against all targeted pathogens [mean±SEM spot forming cells (SFC)/2×105 input cells, with lysate:CMV:211±41; EBV:1137±165; BK:551±109; Asp: 143±8; with peptides: CMV: 251±111; EBV: 1031±238; BK: 616±119 SFC; Asp: 463±132). Given that all donors were CMV and EBV seropositive and BK or Asp prior donor exposure wasnot tested, our data suggest that BK or Aspspecificity can be obtained practically from all healthy individuals, due to the particularly high exposure of the general population to these pathogens. Notably, the combination of Asp targeted-proteins (Crf1, Gel1 and SHMT) induced stronger Th1 responses compared to Asp lysate (p=0,049). Overall, we established arapid and simple, optimizedprotocol of generating clinically relevant numbers of mp-STs from a small amount of donor blood. Should mp-STs move to the clinic and prove safe and effective, they will be an ideal treatment for patients suffering from life-threatening, post-transplant infections.

Adoptive transfer of rapidly-generated multivirus-specific T cells to treat Adv, EBV, CMV, BK and HHV6 infections of HSCT recipients

Severe and fatal viral infections remain common after HSCT. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for EBV, CMV and Adv antigens can treat infections that are impervious to conventional therapies, but extension to additional viruses has been limited by competition between virus-derived antigens and laborious manufacturing procedures. We are now evaluating whether infusion of rapidly-generated donor-derived pentavalent T cell lines (pCTL), stimulated just once with peptide libraries spanning immunogenic EBV (LMP2, EBNA1, BZLF1), Adv (Hexon, Penton), CMV (pp65, IE1), BK (Large T, VP1) and HHV6 (U90, U11, U14) antigens, and expanded in the presence of IL4+7 in a G-Rex device, is safe and effective in HSCT recipients with active infections. With NHLBI-PACT support, 35 clinical-grade pCTL lines have been generated. From 30x106 PBMCs we prepared a mean of 374x106 cells (range 99-713x106) over 9-11 days. The lines were polyclonal, comprising both CD4+ (57±5%) and CD8+ (35±5%) cells, with specificity for CMV (IE1: 337±141; pp65: 1059±479 SFC/2x105), EBV (LMP2: 175±87; EBNA1: 116±44; BZLF1: 129±88), Adv (Hexon: 446±153; Penton: 317±108), BK (Large T: 130±67; VP1: 231±104) and HHV6 (U90: 66±50; U11: 36±18; U14: 82±21) and no alloreactivity against recipient PHA blasts (mean Cr51 release 1% 20:1 E:T). To date we have infused 10 patients on study; 4 on DL1 (5x106/m2), 4 on DL2 (1x107/m2) and 2 on DL3 (2x107/m2), with no adverse events. Three patients were infused prophylactically (38-43 days post-HSCT), while 7 received the cells as treatment for one or more active infections at 59-139 days post-HSCT. Based on viral load measurements by day 42, the pCTLs were successful in controlling active CMV (1 complete (CR) and 1 partial response (PR)), EBV (2 CRs, including a case of frank PTLD); Adv (1 CR); BK (1 CR, 3 PR) and HHV6 (1 CR) infections. Additionally, 1 patient received cells off study as treatment for widespread rituximab-resistant EBV-PTLD. Post-infusion there was an immediate decline in her EBV viral load with PTLD resolution, coincident with an increase in circulating EBV-specific T cells. However, the profound anti-tumor activity mediated by the rapidly-expanding EBV-directed T cells also produced a transient systemic inflammatory response syndrome in this patient, which was controlled with steroids and anti-TNFR antibody, with no long term adverse effects. In summary, infusion of pCTLs has thus far proven safe and is associated with the appearance of virus-reactive T cells in peripheral blood and subsequent virus clearance.