Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells.

BACKGROUND: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19). RESULTS: SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients. CONCLUSIONS: CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach. TRIAL REGISTRATION: Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036. FUNDING: National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-γ). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor.

T cells can be reprogrammed to redirect specificity to tumor-associated antigens (TAAs) through the enforced expression of chimeric antigen receptors (CARs). The prototypical CAR is a single-chain molecule that docks with TAA expressed on the cell surface and, in contrast to the T-cell receptor complex, recognizes target cells independent of human leukocyte antigen. The bioprocessing to generate CAR(+) T cells has been reduced to clinical practice based on two common steps that are accomplished in compliance with current good manufacturing practice. These are (1) gene transfer to stably integrate the CAR using viral and nonviral approaches and (2) activating the T cells for proliferation by crosslinking CD3 or antigen-driven numeric expansion using activating and propagating cells (AaPCs). Here, we outline our approach to nonviral gene transfer using the Sleeping Beauty system and the selective propagation of CD19-specific CAR(+) T cells on AaPCs.

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.

Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated "D-CAR") upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR(+) T cells for clinical trials. The D-CAR(+) T cells exhibited specificity for ß-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR(+) T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR(+) T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.

Activating and propagating polyclonal gamma delta T cells with broad specificity for malignancies.

PURPOSE: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. EXPERIMENTAL DESIGN: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. RESULTS: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1(neg)Vδ2(neg) subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1(neg)Vδ2(neg)>Vδ2) of survival of mice with ovarian cancer xenografts. CONCLUSIONS: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans.

Clinical application of Sleeping Beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR(1-3). This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10(th) the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application(4-8). Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2(nd) generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats(9-11). To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)(12). In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR(+) T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-21(13). Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells.

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

Adoptive transfer of EBV-specific T cells results in sustained clinical responses in patients with locoregional nasopharyngeal carcinoma.

Patients with recurrent or refractory Epstein Barr Virus (EBV)-positive nasopharyngeal carcinoma (NPC) continue to have poor outcomes. Our earlier Phase I dose escalation clinical study of 10 NPC patients showed that infusion of EBV-specific cytotoxic T cells (EBV-CTLs) was safe and had antitumor activity. To better define the overall response rate and discover whether disease status, EBV-antigen specificity, and/or in vivo expansion of infused EBV-CTLs predicted outcome, we treated 13 additional NPC patients with EBV-CTLs in a fixed-dose, Phase II component of the study. We assessed toxicity, efficacy, specificity, and expansion of infused CTLs for all 23 recurrent/refractory NPC patients treated on this Phase I/II clinical study. At the time of CTL infusion, 8 relapsed NPC patients were in remission and 15 had active disease. No significant toxicity was observed. Of the relapsed patients treated in their second or subsequent remission, 62% (5/8) remain disease free (at 17 to 75 mo), whereas 48.7% (7/15) of those with active disease had a CR/CRu (33.3%) or PR (15.4%). In contrast to locoregional disease, metastatic disease was associated with an increased risk of disease progression (HR: 3.91, P=0.015) and decreased overall survival (HR: 5.55, P=0.022). Neither the specificity of the infused CTLs for particular EBV antigens nor their measurable in vivo expansion discernibly influenced outcome. In conclusion, treatment of patients with relapsed/refractory EBV-positive NPC with EBV-CTLs is safe and can be associated with significant, long-term clinical benefit, particularly for patients with locoregional disease.

Osteoradionecrosis of tympanic bone: reconstruction of outer ear canal with pedicled skin flap, combined with hyperbaric oxygen therapy, in five patients.

OBJECTIVE: To evaluate the results of one-stage surgical repair of the meatal skin defect in patients with long-lasting osteoradionecrosis of the outer ear canal, using a postauricular, inferiorly pedicled skin flap. All patients were also treated with hyperbaric oxygen both pre- and post-operatively. METHODS: A prospective study evaluating the results of a one-stage surgical procedure to repair the meatal skin defect in five patients with osteoradionecrosis of the outer ear canal. All patients were treated with hyperbaric oxygen both pre- and post-operatively. RESULTS: In four of the five patients, intact canal skin was achieved after surgery and hyperbaric oxygen therapy. One patient needed a second operation to cover a small remaining area of bare bone. In one patient, wound healing was unsatisfactory and an area of bare bone remained. CONCLUSION: In cases of osteoradionecrosis of the outer ear canal, the skin defect can be repaired with an inferiorly pedicled skin flap. Although not yet scientifically proven, the peri-operative application of hyperbaric oxygen may be of additional value to improve wound healing in areas of compromised tissue.

Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients.

Treatment of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of autologous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at (http://www.clinialtrials.gov) as NCT00608257.

Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma.

Cytotoxic T lymphocytes (CTLs) directed to nonviral tumor-associated antigens do not survive long term and have limited antitumor activity in vivo, in part because such tumor cells typically lack the appropriate costimulatory molecules. We therefore engineered Epstein-Barr virus (EBV)-specific CTLs to express a chimeric antigen receptor directed to the diasialoganglioside GD2, a nonviral tumor-associated antigen expressed by human neuroblastoma cells. We reasoned that these genetically engineered lymphocytes would receive optimal costimulation after engagement of their native receptors, enhancing survival and antitumor activity mediated through their chimeric receptors. Here we show in individuals with neuroblastoma that EBV-specific CTLs expressing a chimeric GD2-specific receptor indeed survive longer than T cells activated by the CD3-specific antibody OKT3 and expressing the same chimeric receptor but lacking virus specificity. Infusion of these genetically modified cells seemed safe and was associated with tumor regression or necrosis in half of the subjects tested. Hence, virus-specific CTLs can be modified to function as tumor-directed effector cells.

The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane.

The Breast Cancer Resistance Protein (BCRP/ABCG2) is a transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. This transporter is highly expressed in many tissues; however, in human kidney, only the mRNA was found in contrast to the mouse kidney, where the transporter is abundant. In bcrp/abcg2((-/-)) mice, the expression of two sterol transporter genes, abcg5 and abcg8, was strongly increased in the kidney, perhaps as a compensatory mechanism to upregulate efflux. We found using immunohistochemical analysis clear localization of BCRP/ABCG2 to the proximal tubule brush border membrane of the human kidney comparable to that of other ABC transporters such as P-glycoprotein/ABCB1, MRP2/ABCC2, and MRP4/ABCC4. Hoechst 33342 dye efflux from primary human proximal tubule cells was significantly reduced by the BCRP/ABCG2 inhibitors fumitremorgin C and nelfinavir. Our study shows that in addition to other apical ABC transporters, BCRP/ABCG2 may be important in renal drug excretion.

P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected against ischemic renal injury.

The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful products of oxidative stress. We characterized renal function of P-gp knockout mice and studied its consequences in renal ischemic damage. Compared with wild-type mice, knockout mice have a lower glomerular filtration rate and renal plasma flow. An augmented urinary excretion of sodium, numerous amino acids, calcium, glucose, and low molecular weight proteins was observed along with an increased diuresis. A higher lithium plasma clearance in the knockout mice suggested proximal tubular dysfunction. Electron microscopy showed mitochondrial abnormalities in proximal tubular cells that could account for decreased adenosine triphosphate levels in the cortex. After inducing ischemia, wild-type mice showed a decrease in creatinine clearance and severe proximal tubular necrosis. In contrast, knockout mice had no signs of tubular damage. Our data indicate that P-gp knockout mice have impaired renal function but are protected against ischemic renal injury.

Complete responses of relapsed lymphoma following genetic modification of tumor-antigen presenting cells and T-lymphocyte transfer.

Epstein-Barr virus (EBV)-associated tumors developing in immunocompetent individuals present a challenge to immunotherapy, since they lack expression of immunodominant viral antigens. However, the tumors consistently express viral proteins including LMP2, which are immunologically "weak" but may nonetheless be targets for immune T cells. We previously showed that a majority of cytotoxic T lymphocytes (CTLs) reactivated using EBV-transformed B-lymphoblastoid cells lines (LCLs) contained minor populations of LMP2-specific T cells and homed to tumor sites. However, they did not produce remissions in patients with bulky disease. We have now used gene transfer into antigen-presenting cells (APCs) to augment the expression and immunogenicity of LMP2. These modified APCs increased the frequency of LMP2-specific CTLs by up to 100-fold compared with unmodified LCL-APCs. The LMP2-specific population expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and 5 of 6 patients with active relapsed disease had a tumor response, which was complete in 4 and sustained for more than 9 months. It is therefore possible to generate immune responses to weak tumor antigens by ex vivo genetic modification of APCs and the CTLs so produced can have substantial antitumor activity. This study is registered at http://www.cancer.gov/clinicaltrials (protocol IDs: BCM-H-9936, NCT00062868, NCT00070226).

Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.

Immunocompromised individuals are at high risk for life-threatening diseases, especially those caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus. Conventional therapeutics are primarily active only against CMV, and resistance is frequent. Adoptive transfer of polyclonal cytotoxic T lymphocytes (CTLs) specific for CMV or EBV seems promising, but it is unclear whether this strategy can be extended to adenovirus, which comprises many serotypes. In addition, the preparation of a specific CTL line for each virus in every eligible individual would be impractical. Here we describe genetic modification of antigen-presenting cell lines to facilitate the production of CD4(+) and CD8(+) T lymphocytes specific for CMV, EBV and several serotypes of adenovirus from a single cell culture. When administered to immunocompromised individuals, the single T lymphocyte line expands into multiple discrete virus-specific populations that supply clinically measurable antiviral activity. Monoculture-derived multispecific CTL infusion could provide a safe and efficient means to restore virus-specific immunity in the immunocompromised host.

In vivo expansion of LMP 1- and 2-specific T-cells in a patient who received donor-derived EBV-specific T-cells after allogeneic stem cell transplantation.

Immunotherapy approaches with antigen-specific cytotoxic T lymphocytes (CTLs) have provided safe and effective prophylaxis and treatment of Epstein-Barr virus (EBV)-associated lymphomas arising after bone marrow transplantation. EBV is also associated with other malignancies including approximately 40% of cases of Hodgkin's disease, making this tumor another potential target for EBV-targeted immunotherapy. This study describes a patient with multiple relapsed EBV positive Hodgkin's Disease who received both autologous and allogeneic EBV CTL lines. After multiple chemotherapeutic and radiotherapy regimens including two autologous stem cell transplants, he received two doses of gene-marked autologous EBV-specific CTL which resulted in disease stabilization for 6 months. The gene-marked EBV-CTL persisted for 12 months in the peripheral blood after which he proceeded to unrelated donor stem cell transplant followed by immunotherapy with donor-derived EBV-specific CTL. Despite low levels of donor chimerism, the patient remains in complete remission 5 years post-allogeneic SCT. Comparison of the autologous and the donor-derived CTL lines showed that the donor line had specificity for two tumor-associated EBV antigens, latent membrane protein (LMP)1 and 2 compared to the autologous line, which only had specificity for LMP2 epitopes. Following infusion of the donor-derived CTL, functional analyses showed that T-cells reactive with both LMP1 and LMP2 epitopes expanded in the peripheral blood, suggesting that strategies to increase their frequency may result in a broader cytotoxic response against EBV+ Hodgkin tumors.

Slc11a1 (formerly Nramp1) polymorphisms and susceptibility to post-transplant lymphoproliferative disease following pediatric liver transplantation.

BACKGROUND: Polymorphisms of the solute carrier family 11 member 1 (Slc11a1) gene have previously been associated with susceptibility to infectious disease, anti-tumor defenses, and autoimmune diseases. We postulated that polymorphisms of the gene may also be associated with susceptibility to post-transplant lymphoproliferative disease (PTLD), a disease thought to be related to an impaired immune response to Epstein-Barr virus (EBV) in immunosuppressed patients. METHODS: Whole blood samples were obtained from 45 pediatric patients who underwent liver transplantation. Polymerase chain reaction (PCR) was used to amplify a 3' region of the gene that includes an exon 15 single-nucleotide substitution (referred to as D543N) and a 4-bp deletion polymorphism (referred to as 3'-UTR). PCR products were digested using AvaII and FokI restriction enzymes for the D543N and 3'-UTR polymorphisms, respectively. PTLD disease status and EBV virus serum titers of all patients were obtained from hospital records. RESULTS: Six of the 45 pediatric transplant recipients developed PTLD. An association was found between 3'-UTR polymorphisms of Slc11a1 and incidence of PTLD after liver transplantation (P = 0.005). In addition, post-transplant serum EBV titers were higher (P = 0.009) for recipients with certain Slc11a1 polymorphisms. No association was found between the D543N polymorphism and incidence of PTLD. CONCLUSION: 3'-UTR polymorphisms of the Slc11a1 gene appear to be associated with susceptibility to PTLD and the immune response to EBV in pediatric liver transplant recipients. Genotyping of pediatric patients undergoing liver transplantation may enable early identification of patients at high risk for developing high EBV titers and/or PTLD.

ABC transporter expression profiling after ischemic reperfusion injury in mouse kidney.

Renal ATP binding cassette (ABC) transporters have an important role in the elimination of metabolic waste products and compounds foreign to the body. The kidney has the ability to tightly control the expression of these efflux transporters to maintain homeostasis, and as a major mechanism of adaptation to environmental stress. In the present study, we investigated the expression of 45 ABC transporter genes in the mouse kidney under basal conditions, after induction of ischemia and after regeneration. Two days after clamping, mice showed a 76% decrease in renal creatinine clearance, which improved clearly within 7 days. This was confirmed by histological examinations. Seven days after ischemia, real-time quantitative Polymerase chain reaction data showed that transcript abundance of abcb1, abcb11, and abcc4 was increased, and that of abca3, abcc2, and abcg2 decreased. Expression of all transporters returned to baseline after 14 days, except for abcb11, which was reduced. Abcb11 is the major liver canalicular bile salt export pump. Here we show for the first time expression in the kidney and localization of the transporter to the apical membrane of proximal tubules. The presence of another novel renal transporter, abca3, was confirmed by Western blotting. Immunohistochemistry showed that abca3 is localized to the peritubular capillaries and apical membrane of proximal tubules. In conclusion, after inducing ischemic reperfusion injury in the kidney, ABC transporters appear to be differentially regulated, which might be associated with the renal regeneration process. Furthermore, we showed for the first time expression and subcellular localization of abcb11 and abca3 in mouse kidney.