Björn Carlsson

Ex vivo stimulation of cytomegalovirus (CMV)‐specific T cells using CMV pp65‐modified dendritic cells as stimulators

Summary. Cytomegalovirus (CMV) infection is a dangerous complication in immunosuppressed individuals such as allogeneic stem cell transplant patients. CMV disease can be prevented by the early post‐transplant transfer of donor‐derived, CMV‐directed, T cells. Fast and cost efficient methods to generate CMV‐specific T cells are, therefore, warranted. The current study utilized peptide‐pulsed and adenovirus‐transduced dendritic cells (DC) to generate CMV‐restricted T cells. After one stimulation with CMV pp65495−503 peptide‐pulsed DC and three re‐stimulations with peptide‐pulsed monocytes, virtually all T cells were CD8+, expressed the relevant T cell receptor and exhibited high peptide‐specific lytic activity. After only one stimulation, pp65495−503‐restricted T cells could be sorted to a purity of higher than 95% and expanded up to 1000‐fold in 2u2003weeks. This technique may prove useful for the rapid generation of large quantities of specific cytolytic T lymphocytes (CTL) for cell therapy. DC transduced with an adenoviral vector encoding the full‐length pp65 protein (Adpp65) were able to simultaneously expand CTL against multiple epitopes of pp65. In addition, they activated CMV‐specific CD4+ T‐helper cells. This approach would stimulate multiple‐epitope populations of pp65‐specific T cells and could be made available to patients of any human leucocyte antigen (HLA) haplotype. DC transduced with adenoviral vectors to express full‐length antigens may prove to be potent vaccines against viral pathogens and cancer.

Efficient Adenovector CD40 Ligand Immunotherapy of Canine Malignant Melanoma

Cutaneous canine melanomas are usually benign in contrast to human malignant melanoma. However, the canine oropharyngeal, uveal, and mucocutaneous neoplasms are aggressive and have metastatic potential. Surgery and to a lesser extent radiotherapy and chemotherapy are widely adopted treatments but are seldom curative in advanced stages. The similarities between human and canine melanoma make spontaneous canine melanoma an excellent disease model for exploring novel therapies. Herein, we report the first 2 adenovector CD40L immunogene (AdCD40L) treatments of aggressive canine malignant melanoma. Case no. 1 was an advanced stage III oral melanoma that was cured from malignant melanoma with 2 intratumor AdCD40L injections before cytoreductive surgery. After treatment, the tumor tissue was infiltrated with T lymphocytes and B lymphocytes suggesting immune activation. This dog survived 401 days after the first round of gene therapy and was free of melanoma at autopsy. Case no. 2 had a conjunctival malignant melanoma with a rapid progression. This case was treated with 6 AdCD40L injections over 60 days. One hundred and twenty days after start of gene therapy and 60 days after the last injection, the tumor had regressed dramatically, and the dog had a minimal tumor mass and no signs of progression or metastasis. Our results indicate that AdCD40L immunogene therapy is beneficial in canine malignant melanoma and could be considered for human malignant melanoma as well.

Adoptive T-cell therapy for malignant melanoma patients with TILs obtained by ultrasound-guided needle biopsy

Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) can mediate objective responses in up to 50% of malignant melanoma patients with a good performance status refractory to standard treatments. Current protocols for generation of TILs rely on open surgery for access to tumor tissue. We obtained tumor material by ultrasound-guided core needle biopsy or surgery from melanoma patients with progressive disease and were able to isolate >5xa0×xa0106 TILs from 23 of 24 patients who were subsequently treated with these cells. One-third of the individual TIL-positive cultures displayed interferon gamma activity after stimulation with relevant melanoma cell lines. When expanded TILs were used for treatment in combination with daily low dose s.c. IL-2 after prior lymphodepleting chemotherapy, we observed objective clinical responses in one patient treated with TILs obtained from surgery and 4 patients treated with TILs from core biopsies. The results of this study demonstrate for the first time the potential of core biopsies for generation of relevant numbers of TILs that can mediate objective responses in patients with metastatic malignant melanoma. Ultrasound-guided core needle biopsy is a robust, safe and inexpensive approach to obtain tumor tissue for TIL generation, and is especially valuable in instances where surgery is contraindicated.

Impact of surface waves in a Regional Climate Model

A coupled regional atmosphere-wave model system is developed with the purpose of investigating the impact of climate changes on the wave field, as well as feed-back effects of the wave field on the atmospheric parameters. This study focuses on the effects of introducing a two-way atmosphere-wave coupling on the atmosphere as well as on wave parameters. The model components are the regional climate model RCA, and the third generation wave model WAM. Two different methods are used for the coupling, using the roughness length and only including the effect of growing sea, and using the wave age and introducing the reduction of roughness due to decaying sea (swell). Introducing a two-way coupling results in an altered frequency distribution of wind speed and wave heights. When only including growing sea the impact of waves on the long term mean atmospheric parameters is limited, inducing a reduction of wind speed and significant wave height. When also the impact of swell is introduced, there is a shift towards higher wind speeds as well as higher significant wave heights in the four investigated areas. There is a reduction of surface heat fluxes and a decrease in near surface temperature as well as a significant increase in near surface humidity. The major conclusion is that when introducing a more realistic surface description over sea, the air-sea interaction represented by waves has a significant impact also on long term averages of parameters in the atmosphere. Waves should thus be introduced in climate models for a realistic description of processes over sea.

Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials

Adoptive transfer of regulatory T cells (Tregs) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with Treg therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4+CD25highCD127low Tregs from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare Treg preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded Treg preparations have been characterized by their purity, cytokine production and in‐vitro suppressive ability. The results show that Treg preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the Treg preparations. In particular, FACS‐sorted Treg preparations expanded with mature DCs secrete more interleukin (IL)‐10 and granzyme B than FACS‐sorted Treg preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with Tregs from uraemic patients that may guide future efforts to produce clinical‐grade Tregs for use in kidney transplantation.

High Frequency of Prostate Antigen-Directed T Cells in Cancer Patients Compared to Healthy Age-Matched Individuals

In order to obtain a sustained cytotoxic T lymphocyte (CTL) response against cancer cells it is preferable to have CTLs directed against multiple peptide epitopes from numerous tumor‐associated antigens.

Photochemical pathogen inactivation of human serum enables its large-scale application in clinical cell transplantation

Stringent protocols for cGMP, issued by regulatorybodies in Asia, Europe and North America, regulateproduction of cells and tissues for clinical therapy. Dueto the risk of transferring infections, human serum, thepreferred supplement throughout the process of cell cul-ture and transplantation, cannot be used, resulting ininferior quality of released cell products. Photochemicaltreatment (INTERCEPT Cerus Europe BV, Amersfoort,Netherlands), with the psoralen compound, amotosalenHCl and long-wavelength ultraviolet light) has beenshown to inactivate any pathogen of RNA and DNAorigin [1–4]. INTERCEPT treatment of human serumcould allow its use in processing cells and tissues forclinical application.No long-term exposure in vitro of cells to residual amo-tosalen has been performed. However, a carcinogenicitystudy in p53 knockout mice exposed to doses 1000-foldthe clinical dose for 26 weeks, showed no evidence ofcarcogenicity [5].The use of PI-HS compared with C-HS was examined onhuman melanoma cell lines as well as in the production ofclinical grade islets of Langerhans (for the treatment ofType I Diabetes), mesenchymal stem cells (for the MSCs:treatment of GvHD grade III or IV) and T cells (for immunetherapy against malignant melanoma).There was no difference in rate of proliferation during a3-day culture in medium supplemented with either PI-HSor C-HS (10%) for five human melanoma cell-lines(397mel, 526mel, SK23mel, EB81mel, CP64mel).Similarly, there was no difference between islets culturedfor 4 days inCMRL-1066 supplemented with PI-HS orC-HSintermsofinsulinstimulationindexfromadynamicperifu-sion system where the average of the high values dividedwith the average of the low values (14AE7±4AE15, 10AE0±4AE57), the ADP⁄ATP-ratio (0AE010 ± 0AE013, )0AE0004 ±0AE013) and the capacity to cure STZ-diabetic mice (78%,87%). Likewise, no difference was found in intracellularinsulin content (2AE6±1AE14, 2AE3±0AE85 ng⁄ml), expressionof IL-6 (0AE0067 ± 0AE016, 0AE0075 ± 0AE018 pmol⁄lgDNA),IL-8 (0AE264 ± 0AE150, 0AE352 ± 0AE169 pmol⁄lgDNA), MCP-1(0AE164 ± 0AE086, 0AE166 ± 0AE080 pmol⁄lgDNA) or TissueFactor(0AE034 ± 0AE007,0AE087 ± 0AE057 pmol⁄lgDNA).Also, expansion of MSC and the capacity for immuno-suppression in MLC and PHA stimulated lymphocyte cul-tures (71 ± 13, 59 ± 9%) did not differ between MSCgenerated in CMRL-1066 supplemented with PI-HS orC-HS.Finally, no differences in regards of total number of Tcells generated (30AE9±3AE1, 29AE7±1AE5 millions⁄ml), foldexpansion (309AE4±31AE8, 297AE4±15AE2) or expression ofCD25, CD27 or CD26L were observed when T cells were cul-tured in RPMI-1640 supplemented with PI-HS or C-HS.Students t-test was used to evaluate the results between PIand control.The presented technique for PI of human serum providesa solution to an important safety and regulatory problem inmodern cell therapy. PI exerts no negative impact onhuman islets of Langerhans, MSCs, T cells or cell lines andcan even have a positive effect by down-regulation ofinflammatory mediators induced by DNA or RNA strandsreleased from damaged cells. INTERCEPT treatment ofhuman serum allows the routine use of human serum inclinical cell transplantation.In many European countries, the Intercept technology isroutinely used for PI of platelets and plasma for clinicaluse. Applying this already used technique in a new waycould be a new niche for blood banks. They can use this toprovide safer products for clinical cell culture and trans-plantation and by doing this increase the safety for thepatients.

Strategic use of an adenoviral vector for rapid and efficient ex vivo-generation of cytomegalovirus pp65-reactive cytolytic and helper T cells

Cytomegalovirus (CMV) reactivation can cause severe complications for transplant patients. Such patients can be protected against CMV‐associated diseases through reconstitution of donor‐derived CMV‐reactive cytolytic and helper T cells. We have developed a strategic protocol for efficient simultaneous generation of CMV‐reactive CD8+ and CD4+ T cells ex vivo. The protocol uses peripheral blood lymphocytes (PBLs), antigen‐modified mature dendritic cells (DCs) generated in only 3u2003d and an adenoviral vector encoding the CMV pp65 antigen (Adpp65) both as an endogenous and exogenous source of antigen. PBLs stimulated once with Adpp65‐transduced DCs (endogenously expressed pp65) resulted in preferential activation and expansion of pp65‐specific CD8+ T cells while PBLs stimulated with DCs pulsed with cell lysate from Adpp65‐transduced autologous monocytes (exogenously expressed pp65) yielded pp65‐specific CD4+ T cells. Stimulation with double‐modified DCs efficiently activated and expanded cytolytic and helper T cells simultaneously. The frequency of T cells producing interferon‐γ in response to pp65 increased after one stimulation on average 9·6‐fold to 4·3% for CD8+ T cells and 25·8‐fold to 6·5% for CD4+ T cells. This implies that sufficient number of pp65‐specific cytolytic and helper T cells for adoptive transfer may be obtained in only 2u2003weeks.

Distribution of adoptively transferred porcine T-lymphoblasts tracked by 18F-2-fluoro-2-deoxy-d-glucose and position emission tomography

INTRODUCTIONnAutologous or allogeneic transfer of tumor-infiltrating T-lymphocytes is a promising treatment for metastatic cancers, but a major concern is the difficulty in evaluating cell trafficking and distribution in adoptive cell therapy. This study presents a method of tracking transfusion of T-lymphoblasts in a porcine model by (18)F-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG) and positron emission tomography.nnnMETHODSnT-lymphoblasts were labeled with the positron-emitting tracer [(18)F]FDG through incubation. The T-lymphoblasts were administered into the bloodstream, and the distribution was followed by positron emission tomography for 120 min. The cells were administered either intravenously into the internal jugular vein (n=5) or intraarterially into the ascending aorta (n=1). Two of the pigs given intravenous administration were pretreated with low-molecular-weight dextran sulphate.nnnRESULTSnThe cellular kinetics and distribution were readily quantifiable for up to 120 min. High (78.6% of the administered cells) heterogeneous pulmonary uptake was found after completed intravenous transfusion. The pulmonary uptake was decreased either by preincubating and coadministrating the T-lymphoblasts with low-molecular-weight dextran sulphate or by administrating them intraarterially.nnnCONCLUSIONSnThe present work shows the feasibility of quantitatively monitoring and evaluating cell trafficking and distribution following administration of [(18)F]FDG-labeled T-lymphoblasts. The protocol can potentially be transferred to the clinical setting with few modifications.

Effector T Cell Analysis of Melanoma Tumor-infiltrating Lymphocyte Cultures Using HLA-ABC Semimatched Melanoma Cell Lines

The generation of T cells with specific reactivity against tumor-associated antigens is prerequisite for adoptive transfer therapy. Melanoma-specific lymphocyte cultures can be established from tumor-infiltrating lymphocytes (TILs) by in vitro culture with high levels of interleukin-2. In this report, we present TIL data originating from 728 attempted cultures from 33 consecutive melanoma biopsy specimens originating from 30 patients. Cultures were analyzed for the presence of interferon gamma (IFNγ)-producing cells upon stimulation with a panel of HLA-ABC semimatched melanoma cell lines. We sought to find whether such cell lines could be used to analyze TIL reactivity. Cell lines were used as stimulators to circumvent the need for autologous primary tumor cells. Melanoma-reactive cultures were identified by flow cytometry in 25 of the 30 patients. Four hundred forty-four of 728 (60.9%) cultures contained TILs at the end of experiment. Ninety-one of 318 cultures (28.6%) contained IFNγ-producing cells after stimulation. In HLA-A*0201+ patients IFNγ analysis was complemented with melanoma-specific tetramer staining. All but one HLA-A*0201+ patient had MART-1/Melan-A27-35-directed TILs, with frequencies ranging from 0.1% to 90% of CD8+ cells. In addition, tetramer analysis also identified TILs directed against gp100, Tyrosinase, and Her2Neu. Tumor material was collected via needle biopsy in 16 cases and surgery in 18 cases. Overall, surgical material generated more cultures positive for T cells. The described methods are efficient in characterizing clinically relevant melanoma-reactive TILs.