CD8 T cell tolerance to a tumor-associated self-antigen is reversed by CD4 T cells engineered to express the same T cell receptor.

Ag receptors used for cancer immunotherapy are often directed against tumor-associated Ags also expressed in normal tissues. Targeting of such Ags can result in unwanted autoimmune attack of normal tissues or induction of tolerance in therapeutic T cells. We used a murine model to study the phenotype and function of T cells redirected against the murine double minute protein 2 (MDM2), a tumor-associated Ag that shows low expression in many normal tissues. Transfer of MDM2-TCR-engineered T cells into bone marrow chimeric mice revealed that Ag recognition in hematopoietic tissues maintained T cell function, whereas presentation of MDM2 in nonhematopoietic tissues caused reduced effector function. TCR-engineered CD8(+) T cells underwent rapid turnover, downmodulated CD8 expression, and lost cytotoxic function. We found that MDM2-TCR-engineered CD4(+) T cells provided help and restored cytotoxic function of CD8(+) T cells bearing the same TCR. Although the introduction of the CD8 coreceptor enhanced the ability of CD4(+) T cells to recognize MDM2 in vitro, the improved self-antigen recognition abolished their ability to provide helper function in vivo. The data indicate that the same class I-restricted TCR responsible for Ag recognition and tolerance induction in CD8(+) T cells can, in the absence of the CD8 coreceptor, elicit CD4 T cell help and partially reverse tolerance. Thus MHC class I-restricted CD4(+) T cells may enhance the efficacy of therapeutic TCR-engineered CD8(+) T cells and can be readily generated with the same TCR.

Expression of a dominant T-cell receptor can reduce toxicity and enhance tumor protection of allogeneic T-cell therapy.

Due to the lack of specificity for tumor antigens, allogeneic T-cell therapy is associated with graft-versus-host disease. Enhancing the anti-tumor specificity while reducing the graft-versus-host disease risk of allogeneic T cells has remained a research focus. In this study, we demonstrate that the introduction of 'dominant' T-cell receptors into primary murine T cells can suppress the expression of endogenous T-cell receptors in a large proportion of the gene-modified T cells. Adoptive transfer of allogeneic T cells expressing a 'dominant' T-cell receptor significantly reduced the graft-versus-host toxicity in recipient mice. Using two bone marrow transplant models, enhanced anti-tumor activity was observed in the presence of reduced graft-versus-host disease. However, although transfer of T-cell receptor gene-modified allogeneic T cells resulted in the elimination of antigen-positive tumor cells and improved the survival of treated mice, it was associated with accumulation of T cells expressing endogenous T-cell receptors and the development of delayed graft-versus-host disease. The in-vivo deletion of the engineered T cells, mediated by endogenous mouse mammary tumor virus MTV8 and MTV9, abolished graft-versus-host disease while retaining significant anti-tumor activity of adoptively transferred T cells. Together, this study shows that the in-vitro selection of allogeneic T cells expressing high levels of a 'dominant' T-cell receptor can lower acute graft-versus-host disease and enhance anti-tumor activity of adoptive cell therapy, while the in-vivo outgrowth of T cells expressing endogenous T-cell receptors remains a risk factor for the delayed onset of graft-versus-host disease.

Improved monitoring of ovarian stimulation using 3D transvaginal ultrasound plus automated volume count.

Two-dimensional transvaginal ultrasound (2D) is typically performed to monitor follicle growth in IVF and to determine the optimal time for administering human chorionic gonadotrophin. However, 2D only provides an approximation of the real volume of follicles and therefore cannot be used to guarantee standards for follicular measurement. The automated measurement of follicular size in three dimensions (3D) using a software programme that identifies and quantifies hypoechoic regions within a 3D dataset might provide an objective, fast, valid and reliable standard for such measurements. A prospective controlled study (group I: 20 patients, 2D; group II: 20 patients, 3D) investigated how the criteria for triggering oocyte maturation that are normally used in 2D compare to the new and more accurate method of measuring follicles using 3D-based automated volume count. Significantly more oocytes were fertilized (group 1: 7.1 +/- 4.5, group 2: 11.5 +/- 6.4; P < 0.03) when using 3D technology and automated volume count. The study assumes that the automated volume count more closely mirrors the biological reality, which means that it can also be used to guarantee the quality standards established by the European Union directive on tissues and cells (2004/23/EC). This new technology therefore holds great promise of becoming the new standard for monitoring follicular growth in IVF.

Non-informative results and monosomies in PGD: the importance of a third round of re-hybridization.

The incidence of non-informative results after fluorescence in-situ hybridization (FISH) was analysed in preimplantation genetic diagnosis (PGD). FISH was performed on seven chromosomes (13, 16, 18, 21, 22, X, and Y) in two rounds of hybridization (one biopsied blastomere per day 3 embryo). A third round with telomeric probes was performed in order to analyse the chromosome(s) in question. A total of 702 embryos out of a total of 719 embryos from 95 cycles were analysed. The remaining 17 embryos were anucleated and/or had poor quality and could not be diagnosed. After FISH analysis, 52.7% of blastomeres were found to be abnormal, 27.1% euploid, and 20.2% had non-informative results. Abnormalities considered as non-informative included 'monosomy in question' (46.5%), 'trisomy in question' (40.2%), compound aneuploidy (8.5%), and 'no result' (4.9%) for a tested chromosome. Following re-hybridization with telomeric probes, euploidy was found in 42.4% of 'monosomies in question,' in 82.4% of 'trisomies in question,' in 16.7% of compound aneuploidies, and in 71.4% of 'no results' for a tested chromosome. Only 4.2% of non-informative results could not be rescued. This study clearly demonstrates the importance of re-hybridizing non-informative results and monosomies using a third round of hybridization with telomeric probes for chromosome(s) in question.

Redirection to the bone marrow improves T cell persistence and antitumor functions.

A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.

T cell tuning for tumour therapy: Enhancing effector function and memory potential of therapeutic T cells.

The genetic engineering of T cells can lead to enhanced immune-mediated tumour destruction and harbors a great potential for the treatment of cancer. Recent efforts have centered on the design of receptors to re-direct the specificity of T cells towards tumour antigens by means of viral gene transfer. This strategy has shown great success in a number of phase one clinical trials. However, there are still challenges to overcome. On the one hand, T cell function can be further improved to optimize the therapeutic outcome. On the other hand, so called safety switches are required to deal with possible on and off target toxicities. In this review, we will give a brief summary of the success and risks of T cell gene therapy before discussing in detail current strategies to enhance effector function, persistence and safety of adoptively transferred T cells.

Genetic Regulation of Fate Decisions in Therapeutic T Cells to Enhance Tumor Protection and Memory Formation.

A key challenge in the field of T-cell immunotherapy for cancer is creating a suitable platform for promoting differentiation of effector cells while at the same time enabling self-renewal needed for long-term memory. Although transfer of less differentiated memory T cells increases efficacy through greater expansion and persistence in vivo, the capacity of such cells to sustain effector functions within immunosuppressive tumor microenvironments may still be limiting. We have therefore directly compared the impact of effector versus memory differentiation of therapeutic T cells in tumor-bearing mice by introducing molecular switches that regulate cell fate decisions via mTOR. Ectopic expression of RAS homolog enriched in brain (RHEB) increased mTORC1 signaling, promoted a switch to aerobic glycolysis, and increased expansion of effector T cells. By rapidly infiltrating tumors, RHEB-transduced T cells significantly reduced the emergence of immunoedited escape variants. In contrast, expression of proline-rich Akt substrate of 40 kDa (PRAS40) inhibited mTORC1, promoted quiescence, and blocked tumor infiltration. Fate mapping studies following transient expression of PRAS40 demonstrated that mTORC1(low) T cells made no contribution to initial tumor control but instead survived to become memory cells proficient in generating recall immunity. Our data support the design of translational strategies for generating heterogeneous T-cell immunity against cancer, with the appropriate balance between promoting effector differentiation and self-renewal. Unlike pharmacologic inhibitors, the genetic approach described here allows for upregulation as well as inhibition of the mTORC1 pathway and is highly selective for the therapeutic T cells without affecting systemic mTORC1 functions.

Outpatient follicle monitoring: a plea for standardization in ultrasound based follicle monitoring and data transfer.

BACKGROUND: The complexity of assisted reproductive technology (ART) increased during the last decades. New scientific and medical findings as well as the statutory requirements for improving the safety and the outcome of ART were the main impetus for its development. While therapy planning is done and ART is used by the IVF centers, the medical support and monitoring of patients is conducted by referring gynecologists. Reported follicle measurements by the gynecologist allow the adoption of the therapy plan. Most notably, the crucial aspect is processing and interpretation of ultrasound scan (US). The results of the received US, the transfer of data between IVF center(s) and referred physician(s) as well as the subjective interpretation often culminate in interpretation and logistical problems. This might increase the error probability with considerable detriments for the patients and ART outcome. METHODS: The follicle monitoring was performed using Voluson I ultrasound system combined with SonoAVC(®) software. Results were communicated via DICOM language to DynaMed(®) software, a medical program for managing an IVF center with seamless integration of all processes needed for an accurate and precise workflow. RESULTS: In this study, no loss of data was detected. All data were integrated by DynaMed(®) software and were recallable in a fast and easy manner. CONCLUSION: The broad usage of Voluson I ultrasound SonoAVC(®) software and communication of the results via Picture Archiving and Communication System (PACS) server between the IVF center and local gynecologist would provide more assistance for the patients and consequently the ART outcomes can be improved.

Dopamine agonist bromocriptine for the prevention of ovarian hyperstimulation syndrome.

The aim of this retrospective study is to investigate the frequency and severity of ovarian hyperstimulation syndrome and the pregnancy rate in a patient collective at risk who received bromocriptine treatment.

The rationale behind collecting umbilical cord blood.

Umbilical cord blood (UCB) is an increasingly important and rich source of stem cells. These cells can be used for the treatment of many diseases, including cancers and immune and genetic disorders. For patients for whom no suitable related donor is available, this source of hematopoietic stem cells offers substantial advantages, notably the relative ease of procurement, the absence of risk to the donor, the small likelihood of transmitting clinically important infections, the low risk of severe graft-versus-host disease (GVHD) and the rapid availability of placental blood for transplantation centers. Even though almost 80 diseases are treatable with cord blood stem cells, 97 percent of cord blood is still disposed of after birth and lost for patients in need! To improve availability of stem cells to a broader community, efforts should be undertaken to collect cord blood and expectant parents should be properly informed of their options with regard to cord blood banking.