Frequency and functional characterization of fusion genes in squamous cell carcinoma of the lung.

INTRODUCTION: In contrast to lung adenocarcinoma (LUAD), targetable genetic alterations are less frequently detected in squamous cell carcinoma of the lung (LUSC). Over the last years, gene fusions have become promising targets in many solid cancers. Here, we analysed a cohort of LUSC, identified recurrent fusion genes and functionally characterised these tumour genomes. METHODS: A subset of 1608 squamous cell carcinomas of the lung was analysed by means of the FusionPlex® Lung Panel to identify potentially targetable gene fusions using targeted next-generation sequencing. Cases harbouring recurrent gene fusions were further analysed using FISH, Cytoscan HD arrays and cell culture experiments. RESULTS: We found both, known and novel gene fusions in about 3 % of the cases. Known fusions occurring in lung cancer included ALK::EML4, EGFRvIII, EZR::ROS1 and FGFR3::TACC. We further identified recurrent gene fusions of currently unknown biological function, involving EGFR::VSTM2A and NSD3::FGFR1 and showed that the occurrence of the EGFR::VSTM2A fusion is accompanied by high-level amplification of EGFR. Our analyses further revealed that the genomes of these LUSC patients are chromosomally unstable, which leads us to believe that such non-actionable genomic rearrangements may be a result of "chromosomal chaos" most probably not representing exclusive cancer-driving genes in this cancer entity. CONCLUSIONS: We emphasise that caution should be taken when novel fusions are found and that the appearance of new gene fusions should always be interpreted in the molecular context of the respective disease.

IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors.

Chimeric antigen receptor (CAR)-redirected T cell therapy often fails to control tumors in the long term due to selecting cancer cells that downregulated or lost CAR targeted antigen. To reprogram the functional capacities specifically of engineered CAR T cells, we inserted IL12 into the extracellular moiety of a CD28-ζ CAR; both the CAR endodomain and IL12 were functionally active, as indicated by antigen-redirected effector functions and STAT4 phosphorylation, respectively. The IL12-CAR reprogrammed CD8+ T cells toward a so far not recognized natural killer (NK) cell-like signature and a CD94+CD56+CD62Lhigh phenotype closely similar, but not identical, to NK and cytokine induced killer (CIK) cells. In contrast to conventional CAR T cells, IL12-CAR T cells acquired antigen-independent, human leukocyte antigen E (HLA-E) restricted cytotoxic capacities eliminating antigen-negative cancer cells in addition to eliminating cancer cells with CAR cognate antigen. Simultaneous signaling through both the CAR endodomain and IL12 were required for inducing maximal NK-like cytotoxicity; adding IL12 to conventional CAR T cells was not sufficient. Antigen-negative tumors were attacked by IL12-CAR T cells, but not by conventional CAR T cells. Overall, we present a prototype of a new family of CARs that augments tumor recognition and elimination through expanded functional capacities by an appropriate cytokine integrated into the CAR exodomain.

Blocking CD30 on T Cells by a Dual Specific CAR for CD30 and Colon Cancer Antigens Improves the CAR T Cell Response against CD30- Tumors.

Chimeric antigen receptor (CAR)-engineered T cells are efficacious in controlling advanced leukemia and lymphoma, however, they fail in the treatment of solid cancer, which is thought to be due to insufficient T cell activation. We revealed that the immune response of CAR T cells with specificity for carcinoembryonic antigen (CEA) was more efficacious against CEA+ cancer cells when simultaneously incubated with an anti-CD30 immunotoxin or anti-CD30 CAR T cells, although the targeted cancer cells lack CD30. The same effect was achieved when the anti-CD30 single-chain variable fragment (scFv) was integrated into the extracellular domain of the anti-CEA CAR. Improvement in T cell activation was due to interfering with the T cell CD30-CD30L interaction by the antagonistic anti-CD30 scFv HRS3; an agonistic anti-CD30 scFv or targeting the high-affinity interleukin-2 (IL-2) receptor was not effective. T cells with the anti-CD30/CEA CAR showed superior immunity against established CEA+ CD30- tumors in a mouse model. The concept is broadly applicable since anti-CD30/TAG72 CAR T cells also showed improved elimination of TAG72+ CD30- cancer cells. Taken together, targeting CD30 on CAR T cells by the HRS3 scFv within the anti-tumor CAR improves the redirected immune response against solid tumors.

Calcyphosine-like (CAPSL) is regulated in Multiple Symmetric Lipomatosis and is involved in Adipogenesis.

Little is known on the causes and pathogenesis of the adipose tissue disorder (familial) Multiple Symmetric Lipomatosis (MSL). In a four-generation MSL-family, we performed whole exome sequencing (WES) in 3 affected individuals and 1 obligate carrier and identified Calcyphosine-like (CAPSL) as the most promising candidate gene for this family. Screening of 21 independent patients excluded CAPSL coding sequence variants as a common monogenic cause, but using immunohistochemistry we found that CAPSL was down-regulated in adipose tissue not only from the index patient but also in 10 independent sporadic MSL-patients. This suggests that CAPSL is regulated in sporadic MSL irrespective of the underlying genetic/multifactorial cause. Furthermore, we cultivated pre-adipocytes from MSL-patients and generated 3T3-L1-based Capsl knockout and overexpressing cell models showing altered autophagy, adipogenesis, lipogenesis and Sirtuin-1 (SIRT1) expression. CAPSL seems to be involved in adipocyte biology and perturbation of autophagy is a potential mechanism in the pathogenesis of MSL. Downregulation of CAPSL and upregulation of UCP1 were common features in MSL fat while the known MSL genes MFN2 and LIPE did not show consistent alterations. CAPSL immunostainings could serve as first diagnostic tools in MSL clinical care with a potential to improve time to diagnosis and healthcare options.

Construction of human artificial ovary from cryopreserved ovarian tissue: Appearance of apoptosis and necrosis after enzymatic isolation of follicles.

Earlier it was shown that number of retrieved follicles was significantly higher in Tumor Dissociation Enzyme (TDE)-treatment group compare to standard Liberase TM-group. The aim of our present investigations was to examine the effect of TDE on appearance of apoptosis and necrosis in follicles and stromal cells after digesting of cryopreserved ovarian cortex. Fresh and frozen ovarian cortex fragments (OCF) from 14 patients (29 ±â€¯6 years old), sized 20-210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1 frozen OCF digested with TDE; Group 2 frozen OCF digested with LiberaseTM; Group 3 fresh OCF digested with TDE; Group 4 fresh OCF digested with Liberase TM. To differentiate the live, early apoptotic, late apoptotic and necrotic cells in digested ovarian cortex suspension, a flow cytometric apoptosis/necrosis assay with FITC Annexin V Apoptosis Detection Kit and with 7-AAD was performed. Most of fresh (not frozen) cells digested with TDE or Liberase TM (95 ±â€¯2.4% vs. 90.4 ±â€¯3.1%, respectively) as well as in frozen ovarian cortex digested with TDE or Liberase TM (93.1 ±â€¯3.4% vs. 89.7 ±â€¯4.4%, respectively) has located in Q3 quadrant and these cells both negative to 7-AAD and Annexin V were considered as viable. It was established that both types of enzymatic treatment applying to fresh as well as to frozen ovarian cortex resulted to high rate of viable cells (Group 1: 93.8 ±â€¯3.4%; Group 2: 91.8 ±â€¯6.0%; Group 3: 90.5 ±â€¯6.9%; Group 4: 87.3 ±â€¯2.3%) and are non significantly different (P > 0.1) between all treatment groups. The amount of early apoptotic (Group 1: 3.5 ±â€¯1.6%; Group 2: 4.4 ±â€¯1.6%; Group 3: 1.6 ±â€¯1.1%; Group 4: 2.4 ±â€¯1.5%), late apoptotic (Group 1: 2.7 ±â€¯2.4%; Group 2: 44.0 ±â€¯1.9%; Group 3: 3.1 ±â€¯1.1%; Group 4: 2.8 ±â€¯0.7% and necrotic (Group 1: 0.9% ±â€¯0.1%; Group 2: 2.9 ±â€¯0.8%; Group 3: 3.4 ±â€¯4.5%; Group 4: 1.1 ±â€¯0.6%) cells was low and was not significantly different in all treatment groups (P > 0.1). It was concluded that the use of Tumor Dissociation Enzyme, effectiveness of which is higher than Liberase TM, does not lead to increasing of apoptosis and necrosis in follicles and stromal cells after enzymatic digesting of cryopreserved ovarian cortex.

Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex.

BACKGROUND: The aim of this study was to examine the effectiveness of Tumor Dissociation Enzyme (TDE) on the viability of follicles after digestion of fresh and cryopreserved ovarian cortex fragments (OCFs). METHODS: Fresh and thawed OCF from 14 patients (29 ± 6 years), sized 20 to 210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1, frozen OCF digested with TDE; Group 2, frozen OCF digested with LiberaseTM; Group 3, fresh OCF digested with TDE; and Group 4, fresh OCF digested with Liberase TM. Evaluation of follicle viability was performed under light microscope after staining with Neutral red. For visualization of viable and dead cells under a confocal laser scanning microscope, the follicles were stained with Calcein AM and ethidium homodimer-1. RESULTS: The results showed that the number of retrieved follicles was significantly higher (990 vs 487; P < 0.01) in the TDE-treatment group compared to the Liberase TM-group. The presence of intense neutral red stained follicles was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (70.3% ± +/- 6.22 vs 53,1% ± 2.03 and 94.2% ± 6.6 vs 79.1% ± 2.1; P < 0.01). The percentage of Calcein AM stained follicles of class V1 was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (95.97% ± 7.8 vs 87.87% ± 2.4; 97.1% ± 6.8 vs 91.3% ± 2.3; P < 0.01). CONCLUSION: The enzymatic digestion of ovarian cortex with TDE provides recovery of a higher number of healthy preantral follicles in contrast to earlier described Liberase TM procedure.

Role of LTBP4 in alveolarization, angiogenesis, and fibrosis in lungs.

Deficiency of the extracellular matrix protein latent transforming growth factor-ß (TGF-ß)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-ß, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-ß activity resulting in profibrotic processes in the lung.

Temporal and tissue-specific requirements for T-lymphocyte IL-6 signalling in obesity-associated inflammation and insulin resistance.

Low-grade inflammation links obesity to insulin resistance through the activation of tissue-infiltrating immune cells. Interleukin-6 (IL-6) is a crucial regulator of T cells and is increased in obesity. Here we report that classical IL-6 signalling in T cells promotes inflammation and insulin resistance during the first 8 weeks on a high-fat diet (HFD), but becomes dispensable at later stages (after 16 weeks). Mice with T cell-specific deficiency of IL-6 receptor-α (IL-6RαT-KO) exposed to a HFD display improved glucose tolerance, insulin sensitivity and inflammation in liver and EWAT after 8 weeks. However, after 16 weeks, insulin resistance in IL-6RαT-KO epididymal white adipose tissue (EWAT) is comparable to that of controls, whereas the inflammatory profile is significantly worse. This coincided with a shift from classical T cell IL-6 signalling at 8 weeks, to enhanced IL-6 trans-signalling at 16 weeks. Collectively, our studies reveal that IL-6 action in T cells through classical IL-6 signalling promotes inflammation and insulin resistance early during obesity development, which can be compensated for by enhanced IL-6 trans-signalling at later stages.

Antigen-presenting human B cells are expanded in inflammatory conditions.

Traditionally, B cells have been best known for their role as producers of antibodies. However, in recent years, a growing body of evidence has accumulated showing that B cells fulfill a range of other immunologic functions. One of the functions that has attracted increasing attention is the capacity of B cells to induce antigen-specific activation of T cells through presentation of antigens. However, the analysis of this B cell function has been hampered by the lack of a phenotypically well-defined antigen-presenting B cell subset. Here, we report the identification of a human antigen-presenting B cell subset with strong immunostimulatory properties. This B cell subset is characterized by low expression of CD21 and high expression of the activation marker CD86 and exhibits strong T cell-stimulatory activity, as demonstrated by means of an autologous mixed-lymphocyte reaction. Phenotypically, CD21lowCD86pos immunostimulatory B cells (BAPC) represented CD27+ class-switched IgMnegIgDneg B lymphocytes and displayed a higher expression of cell surface receptors, which mediate the migration from peripheral blood to sites of inflammation. Flow cytometric analysis of peripheral blood obtained from individuals with inflammatory conditions revealed that the BAPC subset was expanded following vaccination and in patients with rheumatoid arthritis. Taken together, our work shows that BAPC represents a strongly immunostimulatory B cell subset, which could be a promising target for immunotherapeutic intervention in inflammatory diseases.

Mitofusin 2 is required to maintain mitochondrial coenzyme Q levels.

Mitochondria form a dynamic network within the cell as a result of balanced fusion and fission. Despite the established role of mitofusins (MFN1 and MFN2) in mitochondrial fusion, only MFN2 has been associated with metabolic and neurodegenerative diseases, which suggests that MFN2 is needed to maintain mitochondrial energy metabolism. The molecular basis for the mitochondrial dysfunction encountered in the absence of MFN2 is not understood. Here we show that loss of MFN2 leads to impaired mitochondrial respiration and reduced ATP production, and that this defective oxidative phosphorylation process unexpectedly originates from a depletion of the mitochondrial coenzyme Q pool. Our study unravels an unexpected and novel role for MFN2 in maintenance of the terpenoid biosynthesis pathway, which is necessary for mitochondrial coenzyme Q biosynthesis. The reduced respiratory chain function in cells lacking MFN2 can be partially rescued by coenzyme Q10 supplementation, which suggests a possible therapeutic strategy for patients with diseases caused by mutations in the Mfn2 gene.

Arming cytokine-induced killer cells with chimeric antigen receptors: CD28 outperforms combined CD28-OX40 "super-stimulation".

Cytokine-induced killer (CIK) cells raised interest for use in cellular antitumor therapy due to their capability to recognize and destroy autologous tumor cells in a HLA-independent fashion. The antitumor attack of CIK cells, predominantly consisting of terminally differentiated CD8(+)CD56(+) cells, can be improved by redirecting by a chimeric antigen receptor (CAR) that recognizes the tumor cell and triggers CIK cell activation. The requirements for CIK cell activation were, however, so far less explored and are likely to be different from those of "younger" T cells. We revealed that CD28 and OX40 CARs produced higher interferon- secretion as compared with the first-generation ζ-CAR; CD28-ζ and the third-generation CD28-ζ-OX40 CAR, however, performed similar in modulating most CIK cell effector functions. Compared with the CD28-ζ CAR, however, the CD28-ζ-OX40 CAR accelerated terminal maturation of CD56(+) CIK cells producing high frequencies in activation-induced cell death (AICD) and reduced antitumor efficiency in vivo. Consequently, CD28-ζ CAR CIK cells of CD56(-) phenotype were superior in redirected tumor cell elimination. CAR-mediated CIK cell activation also increased antigen-independent target cell lysis; the CD28-ζ CAR was more efficient than the CD28-ζ-OX40 CAR. Translated into therapeutic strategies, CAR-redirected CIK cells benefit from CD28 costimulation; "super-costimulation" by the CD28-ζ-OX40 CAR, however, performed less in antitumor efficacy due to increased AICD.

Adoptive immunotherapy with redirected T cells produces CCR7- cells that are trapped in the periphery and benefit from combined CD28-OX40 costimulation.

Adoptive therapy of cancer with genetically redirected T cells showed spectacular efficacy in recent trials. A body of preclinical and clinical data indicate that young effector and central memory T cells perform superior in a primary antitumor response; repetitive antigen engagement, however, drives T-cell maturation to terminally differentiated cells associated with the loss of CCR7, which enables T cells to persist in peripheral tissues. In this work, we explored the antitumor efficacy of CCR7(-) T cells when redirected in an antigen-dependent fashion by a chimeric antigen receptor (CAR) toward tumors in the periphery. CAR-engineered CCR7(-) T cells more efficiently accumulated at the tumor site, secreted more IFN-γ, expressed higher amounts of cytotoxic molecules, and showed superior tumor cell lysis compared to the younger CCR7(+) cells. CCR7(-) T cells, however, were more prone to spontaneous and activation-induced cell death, which could be counteracted by simultaneous CD28 and OX40 (CD134) costimulation. Consequently, the combined CD28-ζ-OX40 signaling CAR rescued CCR7(-) T cells from apoptosis, which then produced more efficient antitumor efficacy than CCR7(+) T cells redirected by the same CAR. Data suggest that T-cell therapy will benefit from combined CD28-ζ-OX40 stimulation in the long-term by rescuing continuously generated CCR7(-) T cells for an antitumor attack.

PECAM1(+)/Sca1(+)/CD38(+) vascular cells transform into myofibroblast-like cells in skin wound repair.

Skin injury induces the formation of new blood vessels by activating the vasculature in order to restore tissue homeostasis. Vascular cells may also differentiate into matrix-secreting contractile myofibroblasts to promote wound closure. Here, we characterize a PECAM1(+)/Sca1(+) vascular cell population in mouse skin, which is highly enriched in wounds at the peak of neoangiogenesis and myofibroblast formation. These cells express endothelial and perivascular markers and present the receptor CD38 on their surface. PECAM1(+)/Sca1(+)/CD38(+) cells proliferate upon wounding and could give rise to α-SMA(+) myofibroblast-like cells. CD38 stimulation in immunodeficient mice reduced the wound size at the peak of neoangiogenesis and myofibroblast formation. In humans a corresponding cell population was identified, which was enriched in sprouting vessels of basal cell carcinoma biopsies. The results indicate that PECAM1(+)/Sca1(+)/CD38(+) vascular cells could proliferate and differentiate into myofibroblast-like cells in wound repair. Moreover, CD38 signaling modulates PECAM1(+)/Sca1(+)/CD38(+) cell activation in the healing process implying CD38 as a target for anti-angiogenic therapies in human basal cell carcinoma.

T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice.

BACKGROUND & AIMS: New treatment approaches are needed for patients with pancreatic adenocarcinoma. Carcinoembryonic antigen (CEA) is highly expressed on the surface of pancreatic adenocarcinoma cells; we investigated the effects of cytolytic T cells that recognize CEA in a mouse model of pancreatic carcinoma. METHODS: Immune-competent mice that expressed the CEA transgene (CEAtg) in the intestinal and pulmonary tracts were given intrapancreatic injections of Panc02 CEA(+) cells (express CEA and click beetle luciferase) and tumors were grown for 10 days. Mice were then given single intravenous injections of T cells engineered to express a chimeric antigen receptor (CAR) with high specificity, but moderate affinity, for CEA and a luminescence marker. RESULTS: Injection of the anti-CEA CAR T cells reduced the size of pancreatic tumors to below the limit of detection in all mice and produced long-term tumor eradication in 67% of mice. T cells also eradicated CEA(+) fibrosarcoma cells injected 45 days later. Bioluminescence imaging revealed the accumulation and persistence of the T cells at the tumor site. The efficacy of the T cells did not require lymphodepletion and was not reduced by soluble CEA. Mice developed some noninflammatory infiltrations of CAR(+) T cells in intestine and lung, but there was no evidence of destruction of CEA(+) healthy tissues. CONCLUSIONS: Injection of T cells that target CEA can eradicate tumors grown from CEA(+) pancreatic carcinoma cells in the pancreas of CEAtg mice without autoimmune effects.

The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells.

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.

Regulatory T cells with reduced repressor capacities are extensively amplified in pulmonary sarcoid lesions and sustain granuloma formation.

Sarcoidosis can evolve into a chronic disease with persistent granulomas accompanied by progressive fibrosis. While an unlimited inflammatory response suggests an impaired immune control in sarcoid lesions, it stands in contrast to the massive infiltration with CD4(+)CD25(high)FoxP3(+) regulatory T cells. We here revealed that those Treg cells in affected lung lesions were mainly derived from activated natural Treg cells with GARP (LRRC32)-positive phenotype but exhibited reduced repressor capacities despite high IL-10 and TGF-beta 1 levels. The repressive capacity of blood Treg cells, in contrast, was not impaired compared to age-matched healthy donors. Treg derived cells in granuloma lesions have undergone extensive rounds of amplifications indicated by shortened telomeres compared to blood Treg cells of the same patient. Lesional Treg derived cells moreover secreted pro-inflammatory cytokines including IL-4 which sustains granuloma formation through fibroblast amplification and the activation of mast cells, the latter indicated by the expression of membrane-bound oncostatin M.

CD28 costimulation Impairs the efficacy of a redirected t-cell antitumor attack in the presence of regulatory t cells which can be overcome by preventing Lck activation.

Adoptive T-cell transfer showed promising efficacy in recent trials raising interest in T cells with redirected specificity against tumors. T cells were engineered with a chimeric antigen receptor (CAR) with predefined binding and CD3ζ signaling to initiate T-cell activation. CD28 costimulation provided by a CD28-CD3ζ signaling CAR moreover improved T cell activation and persistence; however, it failed to meet the expectations with respect to mounting attacks against solid tumors infiltrated with regulatory T (Treg) cells. We revealed that a CD28 CAR-redirected T-cell attack is accompanied by higher numbers of Treg cells infiltrating the tumor and is less efficient against cancer cells in presence of Treg cells than a CD3ζ CAR T-cell attack. Deletion of the lck binding moiety in the CD28 CAR endodomain, however, improved redirected anti-tumor activity in presence of Treg cells without impairing interferon-γ (IFN-γ) secretion, proliferation, and cytolysis. CD28 modification abrogated interleukin-2 (IL-2) induction upon CAR engagement which in turn is no longer available to sustain Treg cell persistence. CARs with the modified CD28 endodomain thereby expedite the implementation of adoptive T-cell therapy in patients with a variety of cancer types that are heavily infiltrated by Treg cells.

Gene transfer into human cord blood-derived CD34(+) cells by adeno-associated viral vectors.

OBJECTIVE: Bone marrow-derived CD34(+) cells are currently used in clinical trials in patients with ischemic heart disease. An option to enhance activity of injected progenitors may be offered by genetic engineering of progenitor cells with angiogenic growth factors. Recombinant adeno-associated viral vectors (rAAV) have emerged as a leading gene transfer systems. In contrast to other vector systems in use for genetic engineering of CD34(+) cells, rAAV-mediated gene expression does not depend on vector integration. This is relevant for application in regenerative medicine of ischemic tissues, where transient transgene expression is likely sufficient to achieve therapeutic benefits. MATERIALS AND METHODS: We compared three different human AAV serotypes, packaged as pseudotypes by a helper virus-free production method, for their transduction efficiency in human cord blood-derived CD34(+) cells. We further assessed the impact of vector genome conformation, of alpha(v)beta(5) and alpha(5)beta(1) integrin availability and of the transcription-modulating drugs retinoic acid and Trichostatin A on rAAV-mediated human CD34(+) cell transduction. RESULTS: We provide, for the first time, evidence that hCD34(+) cells can be reproducibly transduced with high efficiency by self-complementary rAAV2 without inducing cytotoxicity or interfering with their differentiation potential. We further show the involvement of alpha(5)beta(1) integrin as a crucial AAV2 internalization receptor and a function for transcription-modulating drugs in enhancing rAAV-mediated transgene expression. CONCLUSION: This study represents a first step toward translation of a combined cellular/rAAV-based therapy of ischemic disease.

Effective proliferation of human regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted by IL-2.

The strength of immune repression by regulatory T (Treg) cells is thought to depend on the efficiency of Treg cell activation. The stimuli and their individual strength required to activate resting human Treg cells, however, have so far not been elucidated in detail. We reveal here that induction of proliferation of human CD4(+)C25(+) Treg cells requires an extraordinary strong CD28 costimulatory signal in addition to TCR/CD3 engagement. CD28 costimulation, noteworthy, cannot be substituted by IL-2 to induce proliferation of Treg cells, which is in contrast to CD4(+)CD25(-) T cells. IL-2, in contrast, prevents spontaneous apoptosis of Treg cells, but does not initiate their amplification. IL-2 and CD28 costimulation clearly exhibit disparate effects on Treg cells which are in contrast to those on CD4(+)CD25(-) T cells. Moreover, the prerequisites for Treg cell proliferation differ strikingly from those for effector T cells, implying a balanced orchestration in initiating and limiting a T cell immune response. In addition, data are of relevance for the design of therapeutic strategies involving IL-2 administration and CD28 costimulation.