David Berglund

Hurdles in therapy with regulatory T cells.

Translating regulatory T cell therapy to the clinic in Europe is making progress but has remaining challenges. Improper activation of the immune system contributes to a variety of clinical conditions, including autoimmune and allergic diseases as well as solid organ and bone marrow transplantation. One approach to counteract this activation is through adoptive therapy with regulatory T cells (Tregs). Efforts to manufacture these cells have led to good maunfacturing practice–compliant protocols, and Treg products are entering early clinical trials. Here, we report the stance of the European Union Cooperation in Science and Technology Action BM1305, “Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies—A FACTT,” which identifies hurdles hindering Treg clinical applications in Europe and provides possible solutions.

Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell–related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group—namely, the tolerant recipients—were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS‐independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross‐validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Screening of mortality in transplant patients using an assay for immune function

BACKGROUND So far, the ImmuKnow Immune Cell Function Assay (Cylex, Inc., Columbia, MD, USA) has been used to assess risks of infection and rejection in transplant patients. We hypothesized that the ImmuKnow assay might be used for mortality screening in transplant patients overall. METHODS In the period of February 2007 to December 2009, at the Uppsala University Hospital, 362 patients who received either kidney, kidney+pancreas, kidney+islet cells, liver or liver+kidney allografts were randomly screened using the ImmuKnow assay. All causes of mortality were compared between two groups: patients with at least one ImmuKnow assay below 175ng/mL and patients with all ImmuKnow assays from 175ng/mL and above. Subsequently, the frequency of rejection within thirty days of the ImmuKnow assay was compared between these two groups. RESULTS The study included 1031 ImmuKnow assays obtained from the 362 patients. A total of 111 patients had at least one ImmuKnow below 175ng/mL and 251 patients had all their ImmuKnow assays from 175ng/mL and above. By January 31st 2010, 16 of 111 patients (14.4%) with at least one ImmuKnow assay below 175ng/mL were deceased, compared to 13 of 251 patients (5.2%) with all ImmuKnow assays from 175ng/mL and above (p=0.0053, Fishers exact test). There was no difference in the frequency of rejection between the two groups (19.8% versus 17.5%, p=0.66). CONCLUSIONS In addition to assessing relative risks of infection and rejection in transplant patients, the ImmuKnow assay may be used to identify patients with increased risk of short-term mortality. Transplant patients being highly overimmunosuppressed as assessed by the ImmuKnow assay do not seem to have a lower risk of short-term rejection.

Functional role of the Ca2+-activated Cl- channel DOG1/TMEM16A in gastrointestinal stromal tumor cells

DOG1, a Ca(2+)-activated Cl(-) channel (CaCC), was identified in 2004 to be robustly expressed in gastrointestinal stromal tumors (GIST). It was rapidly included as a tumor marker in routine diagnostics, but the functional role remained unknown. CaCCs are important regulators of normal physiological functions, but also implicated in tumorigenesis, cancer progression, metastasis, cell migration, apoptosis, proliferation and viability in several malignancies. We therefore investigated whether DOG1 plays a role in the three latter in GIST by utilizing in vitro cell model systems. Confocal microscopy identified different subcellular localizations of DOG1 in imatinib-sensitive and imatinib-resistant cells. Electrophysiological studies confirmed that DOG1-specific pharmacological agents possess potent activating and inhibiting properties. Proliferation assays showed small effects up to 72 h, and flow cytometric analysis of adherent cells with 7-AAD/Annexin V detected no pharmacological effects on viable GIST cells. However, inhibition of DOG1 conveyed pro-apoptotic effects among early apoptotic imatinib-resistant cells. In conclusion, DOG1 generates Cl(-) currents in GIST that can be regulated pharmacologically, with small effects on cell viability and proliferation in vitro. Inhibition of DOG1 might act pro-apoptotic on some early apoptotic GIST cell populations. Further studies are warranted to fully illuminate the function of DOG1 and its potential as therapeutic target.

Increased Inflammatory Response in Cytomegalovirus Seropositive Patients with Alzheimer’s Disease

Alzheimer’s disease (AD) has been associated with increased local inflammation in the affected brain regions, and in some studies also with elevated levels of proinflammatory cytokines in peripheral blood. Cytomegalovirus (CMV) is known to promote a more effector-oriented phenotype in the T-cell compartment, increasing with age. The aim of this study was to investigate the inflammatory response of peripheral blood mononuclear cells (PBMCs) from AD patients and non-demented (ND) controls. Using a multiplex Luminex xMAP assay targeting GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-α, cytokine profiles from PBMCs were analysed after stimulation with anti-CD3/CD28 beads, CMV pp65 peptide mix or amyloid β (Aβ) protofibrils, respectively. CMV seropositive AD subjects presented with higher IFN-γ levels after anti-CD3/CD28 and CMV pp65 but not after Aβ stimulation, compared to CMV seropositive ND controls. When analysing IFN-γ response to anti-CD3/CD28 stimulation on a subgroup level, CMV seropositive AD subjects presented with higher levels compared to both CMV seronegative AD and CMV seropositive ND subjects. Taken together, our data from patients with clinically manifest AD suggest a possible role of CMV as an inflammatory promoter in AD immunology. Further studies of AD patients at earlier stages of disease, could provide better insight into the pathophysiology.

Improving the anti‐inflammatory effect of serum eye drops using allogeneic serum permissive for regulatory T cell induction

To investigate the cytokine composition and anti‐inflammatory effects of allogeneic serum preparations for improved use as serum eye drops.

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.

Evidence for Ca2+-regulated ATP release in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are thought to originate from the electrically active pacemaker cells of the gastrointestinal tract. Despite the presence of synaptic-like vesicles and proteins involved in cell secretion it remains unclear whether GIST cells possess regulated release mechanisms. The GIST tumor cell line GIST882 was used as a model cell system, and stimulus-release coupling was investigated by confocal microscopy of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i), flow cytometry, and luminometric measurements of extracellular ATP. We demonstrate that GIST cells have an intact intracellular Ca(2+)-signaling pathway that regulates ATP release. Cell viability and cell membrane integrity was preserved, excluding ATP leakage due to cell death and suggesting active ATP release. The stimulus-secretion signal transduction is at least partly dependent on Ca(2+) influx since exclusion of extracellular Ca(2+) diminishes the ATP release. We conclude that measurements of ATP release in GISTs may be a useful tool for dissecting the signal transduction pathway, mapping exocytotic components, and possibly for the development and evaluation of drugs. Additionally, release of ATP from GISTs may have importance for tumor tissue homeostasis and immune surveillance escape.

Standardizing the freeze-thaw preparation of growth factors from platelet lysate: STANDARDIZING PLT LYSATE

Over the past decades, the focus on the regenerative properties of platelets (PLTs) has intensified and many PLT‐derived growth factors are readily used in medical settings. A general lack of standardization in the preparation of these growth factors remains, however, and this study therefore examines the dynamics of growth factors throughout the freeze‐thaw procedure.

Single-centre long-term follow-up of live kidney donors demonstrates preserved kidney function but the necessity of a structured lifelong follow-up

Abstract Background. The increase of live kidney donation (LKD) demands that we scrutinize its long-term consequences. Socialized medicine in Sweden has allowed us to survey long-term consequences of LKD with a high response rate. Methods. Between 1974 and 2008, 455 LKDs were performed; 28 donors were deceased and 14 had moved abroad at the time of the survey. Of the remaining 413, 96% agreed to participate in a retrospective study with laboratory testing and answering a questionnaire. Results. Mean age at donation was 49 ± 10 years, and the mean time since nephrectomy was 11 ± 7 years (range 1–33). No death was of renal cause. S-creatinine at follow-up was 93 ± 18 μmol/L, 28% had treated hypertension, of whom only 52% had BP <140/90. Eleven per cent had spot microalbuminuria, and 1% were diagnosed with diabetes mellitus. Seventy-one per cent had check-ups at least every second year, but 14% had no check-ups. Eighty per cent would be willing to donate again if it were possible, and only 3% regretted the donation. Conclusion. Renal function is well preserved in the long term after donation, no case of end-stage renal disease was identified, and a large majority of our donors would donate again if it were possible. Although rates of microalbuminuria and hypertension were at expected levels, a significant number of donors demonstrated elevated blood pressure levels and inadequate antihypertensive treatment. A relatively large number of donors did not receive regular check-ups. Both of these issues demonstrate the need for a better-structured lifelong follow-up.