Advanced T-cell Research Articles

Preparing clinical grade Ag-specific T cells for adoptive immunotherapy trials

The production of clinical-grade T cells for adoptive immunotherapy has evolved from the ex vivo numerical expansion of tumor-infiltrating lymphocytes to sophisticated bioengineering processes often requiring cell selection, genetic modification and other extensive tissue culture manipulations, to produce desired cells with improved therapeutic potential. Advancements in understanding the biology of lymphocyte signaling, activation, homing and sustained in vivo proliferative potential have redefined the strategies used to produce T cells suitable for clinical investigation. When combined with new technical methods in cell processing and culturing, the therapeutic potential of T cells manufactured in academic centers has improved dramatically. Paralleling these technical achievements in cell manufacturing is the development of broadly applied regulatory standards that define the requirements for the clinical implementation of cell products with ever-increasing complexity. In concert with academic facilities operating in compliance with current good manufacturing practice, the prescribing physician can now infuse T cells with a highly selected or endowed phenotype that has been uniformly manufactured according to standard operating procedures and that meets federal guidelines for quality of investigational cell products. In this review we address salient issues related to the technical, immunologic, practical and regulatory aspects of manufacturing these advanced T-cell products for clinical use.

Long-term follow-up of patients with peripheral T-cell lymphomas treated up-front with high-dose chemotherapy followed by autologous stem cell transplantation

We report the results of two prospective phase II studies investigating the role of high-dose sequential chemotherapy, followed by autologous stem cell transplantation (ASCT) in 62 patients with advanced stage peripheral T-cell lymphomas (PTCLs) at diagnosis. Conditioning regimen consisted of mitoxantrone (60 mg/m2) and melphalan (180 mg/m2) or carmustine, etoposide, Ara-C and melphalan followed by peripheral blood stem cell autografting. In an intent-to-treat analysis, 46 out of 62 patients (74%) completed the whole programme, whereas 16 patients did not undergo ASCT, mainly because of disease progression. At a median follow-up of 76 months, the estimated 12-year overall (OS), disease-free and event-free survival (EFS) were 34, 55 and 30%, respectively. OS and EFS were significantly better in patients with anaplastic lymphoma-kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL), as compared with the remaining PTCL. Multivariate analysis showed that patients attaining complete remission (CR) before ASCT had a statistically significant benefit in terms of OS and EFS (P<0.0001). Overall treatment-related mortality rate was 4.8%. In conclusion, our findings indicate (1) up-front high-dose therapy and ASCT are feasible, but could induce a high rate of long-term CR only in patients with ALK-positive ALCL and (2) the achievement of CR before autografting is a strong predictor of better survival.

Tissue microarray analysis of human FRAT1 expression and its correlation with the subcellular localisation of β-catenin in ovarian tumours

The mechanisms involved in the pathogenesis of ovarian cancer are poorly understood, but evidence suggests that aberrant activation of Wnt/β-catenin signalling pathway plays a significant role in this malignancy. However, the molecular defects that contribute to the activation of this pathway have not been elucidated. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) is a candidate for the regulation of cytoplasmic β-catenin. In this study, we developed in situ hybridisation probes to evaluate the presence of FRAT1 and used an anti-β-catenin antibody to evaluate by immunohistochemistry the expression levels and subcellular localisation of β-catenin in ovarian cancer tissue microarrays. Expression of FRAT1 was found in some human normal tissues and 47% of ovarian adenocarcinomas. A total of 46% of ovarian serous adenocarcinomas were positive for FRAT1 expression. Accumulation of β-catenin in the nucleus and/or cytoplasm was observed in 55% ovarian adenocarcinomas and in 59% of serous adenocarcinomas. A significant association was observed in ovarian serous adenocarcinomas between FRAT1 and β-catenin expression (P<0.01). These findings support that Wnt/β-catenin signalling may be aberrantly activated through FRAT1 overexpression in ovarian serous adenocarcinomas. The mechanism behind the overexpression of FRAT1 in ovarian serous adenocarcinomas and its significance is yet to be investigated.

Development of Forodesine Hydrochloride (FH), an Inhibitor of Purine Nucleoside Phosphorylase, for Patients with Chronic Lymphocytic Leukemia (CLL).

Mammalian purine nucleoside phosphorylase (PNP) catalyzes the cleavage of inosine, deoxyinosine, guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. PNP deficiency in humans produces a relatively selective depletion of T cells without much effect on normal B cells. FH, a potent inhibitor of PNP, was designed based on the transition-state analog structure stabilized by the enzyme. Previous studies demonstrated that FH in the presence of dGuo inhibits the proliferation of T-lymphocytes (Kicska et al. PNAS 2001). Based on these observations, we conducted a phase I clinical trial of FH in patients with advanced T-cell malignancies. Significant antileukemic activity was correlated with an increase in plasma FH (median 5 μM) and dGuo (median 14 μM), and an accumulation of intracellular dGuo triphosphate (dGTP) (Gandhi et al, Blood, in press, 2005). High accumulation of dGTP in T-cells may be dependant on activity of deoxynucleoside kinases. Because B-CLL cells have high activity of deoxycytidine kinase, we hypothesized that they would be sensitive to FH. This postulate was tested in primary CLL lymphocytes during in vitro investigations. Lymphocytes from patients with CLL were incubated in vitro with FH (2 μM) in the presence of 10 μM dGuo. Lymphocytes from 3 of 4 patients showed an elevation in the intracellular dGTP levels to a median 30-fold at 8 hr, without any effect on other deoxynucleotides. This increase in dGTP was associated with phosphorylation of p53 at ser15, stabilization of p53, and an increase in p21 protein. The dGTP accumulation was related to induction of apoptosis measured by activation of caspase 8, 9, and 3 and cleavage of PARP. Incubation with either FH or dGuo alone did not result in dGTP accumulation or cell death suggesting that PNP inhibition by FH and phosphorylation of dGuo to dGTP are essential for CLL cell death. Based on these encouraging results, availability of oral formulation and to validate these in vitro data during clinical trial, a phase II study of FH in patients with advanced, fludarabine-refractory CLL has been initiated. FH is administered orally at a dose of 200 mg/day for 7 days each week for 4 weeks (cycle 1). Patients are evaluated after 1 full cycle of therapy and in the absence of serious side effects, or disease progression, they continue the treatment for up to 5 more cycles. Laboratory endpoints such as level of FH and dGuo in plasma, PNP activity, dGTP levels in cells, will be measured and correlated with cytoreduction. It is postulated that a high kinase/low nucleotidase activity leading to the accumulation of intracellular dGTP in target CLL cells and apoptosis (akin to what has been seen in T-cells) will result in response to therapy. This is the first trial of a PNP inhibitor for treatment of B-CLL.

FRAT-2 Preferentially Increases Glycogen Synthase Kinase 3β-mediated Phosphorylation of Primed Sites, Which Results in Enhanced Tau Phosphorylation

Tau is a microtubule-associated protein found primarily in neurons, and its function is regulated by site-specific phosphorylation. Although it is well established that tau is phosphorylated at both primed and unprimed epitopes by glycogen synthase kinase 3 beta (GSK3 beta), how specific proteins that interact with GSK3 beta regulate tau phosphorylation has not been thoroughly examined. Members of the FRAT (frequently rearranged in advanced T-cell lymphoma) protein family have been shown to interact with GSK3 beta, and FRAT-1 has been shown to modulate the activity of GSK3 beta toward tau and other substrates. However, the effects of FRAT-2 on GSK3 beta activity and tau phosphorylation have not been examined. Therefore in this study the effects of FRAT-2 on GSK3 beta activity and tau phosphorylation were examined. In situ, FRAT-2 significantly increased GSK3 beta-mediated phosphorylation of tau at a primed epitope while not significantly affecting the phosphorylation of unprimed sites. Co-immunoprecipitation studies revealed that association of FRAT-2 with GSK3 beta resulted in a significant increase in phosphorylation of a primed substrate but did not alter phosphorylation of an unprimed substrate. Further, in vitro assays using recombinant proteins directly demonstrated that FRAT-2 enhances GSK3 beta-mediated phosphorylation of a primed substrate to a greater extent than an unprimed substrate. In addition, FRAT-2 is phosphorylated by GSK3 beta. This is the first demonstration of a protein differentially regulating the activity of GSK3 beta toward primed and unprimed epitopes.

Microarray analysis of gene regulation in the Hepa1c1c7 cell line following exposure to the DNA methylation inhibitor 5-aza-2 ′-deoxycytidine and 2,3,7,8-tetrachlorodibenzo- p-dioxin

Differential expression of various genes was observed in the Hepa1c1c7 cell line following exposure to the DNA methylation inhibitor 5-aza-2 ′-deoxycytidine (AzaC) and to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). AzaC treatment generally affected genes induced by TCDD by modulating their induction levels. Induction of several genes, such as receptor (calcitonin) activity modifying protein 3 (Ramp3) by TCDD was enhanced by AzaC, although AzaC by itself was without effect. Some genes, such as frequently rearranged in advanced T-cell lymphomas (Frat1), were up-regulated by AzaC alone, with this induction being negatively affected by TCDD. Other genes were induced by AzaC, TCDD and their co-treatment. In contrast, many genes such as small proline-rich protein 1A (Sprr1a) and 2A (Sprr1a) were up-regulated by AzaC, but not significantly affected by TCDD. In addition, a group of genes was down-regulated by AzaC, TCDD and their co-treatment. These findings suggest the TCDD-dependent regulation of various genes to be influenced by cellular DNA methylation status.

Intestinal Lymphoma: Exploration of the Prognostic Factors and the Optimal Treatment

Primary gastrointestinal (GI) lymphoma accounts for 4% to 20% of all non-Hodgkin's lymphomas (NHL), being the most common extranodal site of presentation. However, the optimal management of intestinal lymphoma has not yet been established. During the period of 1994 to 2001, we retrospectively analyzed the clinical features of 67 intestinal lymphoma patients diagnosed according to the Lewin's definitions. The most frequently involved location was ileocecal (35.8%) followed by small bowel (31.3%), large bowel (19.4%), and multiple GI involvement (13.4%). The estimated 5-year overall survival rate was 53%. Out of all treated patients, 49.2% achieved complete response. The advanced stage, poor performance and T-cell phenotype had an adverse prognostic influence on overall survival. In localized diseases (stage 1 and 2), the primary surgical treatment had the most favorable influence on failure-free survival (P < 0.05). The resection of localized intestinal lymphoma may be appropriate as the primary treatment.

Port-A Catheter-associated Nocardia bacteremia Detected by Gallium Inflammation Scan: A Case Report and Literature Review

Central venous catheter-associated Nocardia bacteremia is rarely reported. We present the case of a 48-y-old male with a history of advanced T-cell lymphoma who suffered from recurrent fever and persistent Gram-positive bacillus bacteremia. Port-A catheter-associated Nocardia bacteremia was diagnosed on the basis of the clinical response to removal of the catheter and the finding of increased gallium uptake, along with the Port-A catheter presented in the gallium inflammation scan.

FRAT1 peptide decreases Aβ production in swAPP751 cells

Recently, LiCl has been shown to inhibit amyloid β peptide secretion in association with diminished glycogen synthase kinase β (GSK3β) activity. However, it remains unclear if direct inhibition of GSK3β activity will result in decreased Aβ production. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) protein is a negative regulator of GSK3α/β kinase activity. To examine whether direct inhibition of GSK3α/β kinase activity can lower Aβ production, a FRAT1 peptide was expressed in swAPP751 cells that produce high levels of Aβ. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP751 cells increases both GSK3α and β phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total Aβ and Aβ1-42 was significantly decreased. Thus, we provide evidence that direct regulation of GSK3α/β by FRAT1 peptide significantly decreases Aβ production in swAPP751 cells.

An evaluation of the relationship between the quality of prophylactic cranial radiotherapy in childhood acute leukemia and institutional experience: a Quality Assurance Review Center-Pediatric Oncology Group study

Purpose: The Pediatric Oncology Group Protocol 9404 was a prospective clinical trial of two forms of chemotherapy in childhood T-cell acute lymphoblastic leukemia and advanced stage T-cell lymphoblastic non-Hodgkin’s lymphoma. The protocol called for prophylactic C1 whole brain external beam irradiation, 18 Gy in 2 Gy/fraction for 9 fractions. We hypothesized that a correlation would be found between the number of children irradiated on protocol by an institution and the compliance rate of that institution with radiotherapy quality assurance (QA) guidelines. We also hypothesized that QA compliance would improve as the study progressed.Methods and Materials: We scored the radiation dose as a minor deviation from protocol guidelines if the dose to the prescription point differed from the protocol by 6–10%, and a major deviation if it differed from protocol by > 10%. Treatment volumes were scored as a minor deviation if the margins were less than specified or the fields were excessively large. A major deviation was defined as the transection of a potential leukemia-bearing volume such as would be caused by blocking the cribriform plate, optic nerve, or temporal lobe. When the treating physician submitted a treatment plan and simulator film at the initiation of therapy to the Quality Assurance Review Center (QARC), a rapid turn-around review of the plan and suggestions for improvement was provided. At the end of therapy, all simulator and port films were reviewed at the QARC.Results: We reviewed the data from 353 patients treated at 73 institutions in the United States, Canada, and Europe. Of these patients, 2% (n = 7) were not assessable for QA because of incomplete information. Minor quality deviations were found in 27.7% of patients (n = 98) and major deviations in 7.9% (n = 28). The frequency of major deviations for institutions placing 1–4 patients on study was 11% vs. 5.5% for institutions placing ≥5 patients (p < 0.09). The frequency of minor deviations was 28.6% for institutions placing 1–4 patients on study vs. 27.1% for institutions placing >5 patients (p not significant). The frequency of major deviations fell over time (1996–1997, 15.5% vs. 1998–2001, 4.7%, p < 0.001). The frequency of minor deviations did not (1996–1997, 29.9% vs. 1998–2001, 26.9%, p not significant).Conclusion: For a relatively simple radiotherapy field, with clearly written protocol guidelines and rapid turn-around corrections from QARC, the rate of minor deviations was no different between institutions placing 1–4 patients on study and those placing ≥5. A trend (p < 0.09) was noted, however, for major deviations to decrease as a function of institutional experience, as well as over time (p < 0.001), supporting the validity of the hypothesis that pediatric clinical experience matters in QA for C1 whole brain leukemia radiotherapy.

Characterization and tissue-specific expression of human GSK-3-binding proteins FRAT1 and FRAT2

We have isolated the entire coding sequence of human FRAT2 (frequently rearranged in advanced T-cell lymphomas-2). It exhibits appreciable amino acid identity to FRAT1 (77%) which was initially isolated as frequently being overexpressed in a murine leukemia virus insertion model in murine tumors. FRAT proteins are thought to play a role in Wnt signaling. They can bind to glycogen synthase kinase-3 (GSK-3) and Dishevelled, two proteins involved in Wnt signal transduction. Both hFRAT1 and hFRAT2 are intronless genes localized to the same portion of chromosome 10q24.1 and separated by only 10.7 kb. In a broad range of human tissues FRAT1 and FRAT2 are readily detected and expressed in a near identical pattern. Both species are repressed when the human embryonal carcinoma cell line, NT2/D1, is induced to differentiate with all-trans retinoic acid (RA). This treatment had no appreciable effect on FRAT levels in two other RA-sensitive cell lines that were not of germ cell tumor origin. The overlapping expression patterns suggest these two genes share a regulatory region. Both FRAT genes exhibited three species of mRNA, which varied in representation between tissues. When transiently overexpressed in COS-1 cells, the FRAT proteins were detected in the cytosol and concentrated in the nucleus. Both hFRAT1 and hFRAT2 are implicated in the selective modulation of GSK-3 activity via the Wnt signaling pathway. This study provides a foundation from which to examine the role these proteins play in Wnt-dependent and -independent processes.

Outcome of children with advanced T-cell lymphoid malignancy treated according to a modified POG protocol

Belgaumi, A. F.*; Mustafa, M. M.*; Al-Jefri, A.*; Mahgoub, N.*; Al-Mahr, M.; Al-Musa, A.*; Al-Nasser, A.; Rifai, S.; El-Solh, H. Author Information

A GSK3-binding peptide from FRAT1 selectively inhibits the GSK3-catalysed phosphorylation of Axin and β-catenin

The Axin-dependent phosphorylation of β-catenin catalysed by glycogen synthase kinase-3 (GSK3) is inhibited during embryogenesis. This protects β-catenin against ubiquitin-dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal-ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188–226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3-catalysed phosphorylation of Axin and β-catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a ‘priming’ phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of β-catenin phosphorylation.

Comparison of high-dose therapy and autologous bone marrow transplantation for T-cell and B-cell non-Hodgkin's lymphomas

Advanced T-cell non-Hodgkin's lymphoma in adults has been found to have a poor outlook with conventional chemotherapy. To see if this extends to patients treated with high dose therapy and autologous hematopoietic stem cell transplantation, we reviewed the results with this treatment approach at our institution. From October, 1983, to May, 1988, 41 patients who underwent high-dose therapy and autologous hematopoietic stem cell transplant for recurrent non-Hodgkin's lymphoma were re- biopsied before transplantation to determine their immunophenotype. Seventeen of these patients were found to have a T-cell lymphoma, and 24 had a B-cell lymphoma. All patients were included in the intermediate or high grade non-Hodgkin's lymphoma categories, and none were histologically transformed from a low grade lymphoma. Analysis of the response to autologous transplantation in these two patient populations revealed a slightly better complete response rate for patients with T-cell lymphoma (ie, 59% versus 42%, P = NS). The actuarial 2-year survival was 35% in the T-cell group compared with 30% in the B-cell group (P = NS). The 2-year disease-free survival was 28% for the T-cell and 17% for the B-cell patients. Our results with autologous transplantation for salvage therapy revealed equivalent long- term survival and disease-free survival in both relapsed T- and B-cell non-Hodgkin's lymphoma.

Comparison of High-Dose Therapy and Autologous Bone Marrow Transplantation for T-Cell and B-Cell Non-Hodgkin's Lymphomas

Advanced T-cell non-Hodgkin's lymphoma in adults has been found to have a poor outlook with conventional chemotherapy. To see if this extends to patients treated with high dose therapy and autologous hematopoietic stem cell transplantation, we reviewed the results with this treatment approach at our institution. From October, 1983, to May, 1988, 41 patients who underwent high-dose therapy and autologous hematopoietic stem cell transplant for recurrent non-Hodgkin's lymphoma were re-biopsied before transplantation to determine their immunophenotype. Seventeen of these patients were found to have a T-cell lymphoma, and 24 had a B-cell lymphoma. All patients were included in the intermediate or high grade non-Hodgkin's lymphoma categories, and none were histologically transformed from a low grade lymphoma. Analysis of the response to autologous transplantation in these two patient populations revealed a slightly better complete response rate for patients with T-cell lymphoma (ie, 59% versus 42%, P = NS). The actuarial 2-year survival was 35% in the T-cell group compared with 30% in the B-cell group (P = NS). The 2-year disease-free survival was 28% for the T-cell and 17% for the B-cell patients. Our results with autologous transplantation for salvage therapy revealed equivalent long-term survival and disease-free survival in both relapsed T- and B-cell non-Hodgkin's lymphoma.

Influence of 90Sr, adult thymectomy and antilymphocyte globulin on T-cells in mouse peripheral blood.

Three groups of male CBA mice were treated by (1) 90Sr injection (14.8 kBq/g body weight i.p.), (2) adult thymectomy (ATx) + antilymphocyte globulin (ALG), and (3) 90Sr + ATx + ALG, respectively, and one untreated group served as a control. The relative number of T-lymphocytes was determined in peripheral blood mononuclear cells of individual animals using a dye exclusion cytotoxicity assay with antisera against the T-cell surface membrane marker Thy 1.2 and complement. Total and differential leucocyte counts were also performed. 90Sr irradiation decreased the total number of leucocytes irrespective of type, and the combined treatment of ATx and ALG decreased mainly mononuclear cells and particularly T-cells. The most advanced T-cell depletion in peripheral blood was found in the 90Sr + ATx + ALG treated group with a 97 per cent reduction as compared with untreated controls. ATx + ALG thus proved to be useful for blood T-cell depletion in mice treated simultaneously with 90Sr, and might provide a valuable tool in investigations on the possible role of cell-mediated immune response in radiation-induced oncogenesis, with particular emphasis on selective depletion within the monomorphonuclear compartment.