WT1 TCR gene transfer into haematopoietic stem cells: In vivo functional analysis of WT1-specific T cells

The Wilms tumour antigen is a promising target for T cell-based tumour immunotherapies. Vaccines against WT1 peptides tested in cancer patients showed immunological and molecular responses. However, the clinical responses observed were partial and it is currently not known whether physiological levels of WT1 expression in some healthy tissues results in the deletion or tolerance induction of WT1-specific T cells. In this PhD project, TCR gene transfer into purified haematopoietic stem cells (HSCs) was used to study the thymic development of WT1-specific T cells and their fate in the periphery. Lentiviral constructs containing the genes for an HLAA2 allorestricted, murinised WT1 TCR or the genes for a control, viral peptide-specific, LMP2 TCR, were generated. The conditions for lentivirally-transduced HSC transplants were optimised. The results obtained from WT1 TCR tranduced HSC transplants in HLA-A2Kb transgenic mice demonstrated that thymocytes expressing this high-avidity WT1 TCR were positively selected into CD8 T cells and emerged in the recipient’s periphery. WT1-specific T cells exhibited a memory, CD44hi phenotype correlating with rapid antigen specific killing, proliferation and cytokine secretion of WT1-specific T cells in the absence of vaccination. LMP2-specific T cells exhibited a naive-like, CD44low phenotype without any antigen specific function. WT1-specific T cells persistent long-term in the periphery of transplanted mice, and no autoimmunity was noted. The results presented in this thesis show for the first time that T cell specificity for a tumour-associated, self-antigen did not result in tolerance induction, but instead mediated the spontaneous generation of functionally competent, memory phenotype T cells

Novel thermal annealing methodology for permanent tuning polymer optical fiber Bragg gratings to longer wavelengths

The Bragg wavelength of a polymer optical fiber Bragg grating can be permanently shifted by utilizing the thermal annealing method. In all the reported fiber annealing cases, the authors were able to tune the Bragg wavelength only to shorter wavelengths, since the polymer fiber shrinks in length during the annealing process. This article demonstrates a novel thermal annealing methodology for permanently tuning polymer optical fiber Bragg gratings to any desirable spectral position, including longer wavelengths. Stretching the polymer optical fiber during the annealing process, the period of Bragg grating, which is directly related with the Bragg wavelength, can become permanently longer. The methodology presented in this article can be used to multiplex polymer optical fiber Bragg gratings at any desirable spectral position utilizing only one phase-mask for their photo-inscription, reducing thus their fabrication cost in an industrial setting

High-quality Phase-Shifted Bragg grating sensor inscribed with only one laser pulse in a polymer optical fiber

We present the first phase-shifted polymer optical fiber Bragg grating sensor inscribed with only one KrF laser pulse. The phase shift defect was created directly during the grating inscription process by placing a very narrow blocking aperture, in the center of the UV beam. One laser pulse with a duration of 15 ns and energy 6.3 mJ is adequate to introduce a refractive index change of 0.69×10-4 in the fiber core. The high-quality produced Bragg grating structure rejects 16.3 dB transmitted power, thus providing 97.6% reflectivity, which is well suited for photonic applications. The transmission notch depth is about 10 dB and very sharp notches of 3 dB width ranging from 14 pm is reported. The temperature, strain, and pressure response of the sensor has been characterized showing promising results in applications that require high-precision measurements. The ability to inscribe these high-quality sensors effectively can significantly reduce their production cost in industry

Fast and stable gratings inscription in POFs made of different materials with pulsed 248 nm KrF laser

"© 2018 Optical Society of America. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modifications of the content of this paper are prohibited"[EN] This paper presents fiber Bragg grating (FBG) inscription with a pulsed 248 nm UV KrF laser in polymer optical fibers (POFs) made of different polymers, namely polymethyl methacrylate (PMMA), cyclic-olefin polymer and co-polymer, and Polycarbonate. The inscribed gratings and the corresponding inscription parameters are compared with grating inscribed in POFs made of the aforementioned materials but with the hitherto most used laser for inscription, which is a continuous wave 325 nm UV HeCd laser. Results show a reduction of the inscription time of at least 16 times. The maximum time reduction is more than 130 times. In addition, a reflectivity and a bandwidth close to or higher than the ones with the 325 nm laser were obtained. The polymer optical fiber Bragg gratings (POFBGs) inscribed with the 248 nm laser setup present high stability with small variations in their central wavelength, bandwidth, and reflectivity after 40 days. (c) 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.Fundacao para Ciencia e a Tecnologia (FCT) (SFRH/BPD/109458/2015, UID/EEA/50008/2013).Marques, C.; Min, R.; Leal-Junior, A.; Antunes, P.; Fasano, A.; Woyessa, G.; Nielsen, K.... (2018). Fast and stable gratings inscription in POFs made of different materials with pulsed 248 nm KrF laser. Optics Express. 26(2):2013-2022. https://doi.org/10.1364/OE.26.002013S20132022262Webb, D. J. (2015). Fibre Bragg grating sensors in polymer optical fibres. Measurement Science and Technology, 26(9), 092004. doi:10.1088/0957-0233/26/9/092004Prado, A. R., Leal-Junior, A. G., Marques, C., Leite, S., de Sena, G. L., Machado, L. C., … Pontes, M. J. (2017). Polymethyl methacrylate (PMMA) recycling for the production of optical fiber sensor systems. Optics Express, 25(24), 30051. doi:10.1364/oe.25.030051Hu, X., Saez-Rodriguez, D., Marques, C., Bang, O., Webb, D. J., Mégret, P., & Caucheteur, C. (2015). Polarization effects in polymer FBGs: study and use for transverse force sensing. Optics Express, 23(4), 4581. doi:10.1364/oe.23.004581Pospori, A., Marques, C. A. F., Bang, O., Webb, D. J., & André, P. (2017). Polymer optical fiber Bragg grating inscription with a single UV laser pulse. Optics Express, 25(8), 9028. doi:10.1364/oe.25.009028Marques, C. A. F., Webb, D. J., & Andre, P. (2017). Polymer optical fiber sensors in human life safety. Optical Fiber Technology, 36, 144-154. doi:10.1016/j.yofte.2017.03.010Fasano, A., Woyessa, G., Janting, J., Rasmussen, H. K., & Bang, O. (2017). Solution-Mediated Annealing of Polymer Optical Fiber Bragg Gratings at Room Temperature. IEEE Photonics Technology Letters, 29(8), 687-690. doi:10.1109/lpt.2017.2678481Woyessa, G., Pedersen, J. K. M., Fasano, A., Nielsen, K., Markos, C., Rasmussen, H. K., & Bang, O. (2017). Zeonex-PMMA microstructured polymer optical FBGs for simultaneous humidity and temperature sensing. Optics Letters, 42(6), 1161. doi:10.1364/ol.42.001161Fasano, A., Woyessa, G., Stajanca, P., Markos, C., Stefani, A., Nielsen, K., … Bang, O. (2016). Fabrication and characterization of polycarbonate microstructured polymer optical fibers for high-temperature-resistant fiber Bragg grating strain sensors. Optical Materials Express, 6(2), 649. doi:10.1364/ome.6.000649Woyessa, G., Nielsen, K., Stefani, A., Markos, C., & Bang, O. (2016). Temperature insensitive hysteresis free highly sensitive polymer optical fiber Bragg grating humidity sensor. Optics Express, 24(2), 1206. doi:10.1364/oe.24.001206Leal-Junior, A. G., Frizera, A., & José Pontes, M. (2018). Sensitive zone parameters and curvature radius evaluation for polymer optical fiber curvature sensors. Optics & Laser Technology, 100, 272-281. doi:10.1016/j.optlastec.2017.10.006Stefani, A., Andresen, S., Yuan, W., Herholdt-Rasmussen, N., & Bang, O. (2012). High Sensitivity Polymer Optical Fiber-Bragg-Grating-Based Accelerometer. IEEE Photonics Technology Letters, 24(9), 763-765. doi:10.1109/lpt.2012.2188024Marques, C. A. F., Peng, G.-D., & Webb, D. J. (2015). Highly sensitive liquid level monitoring system utilizing polymer fiber Bragg gratings. Optics Express, 23(5), 6058. doi:10.1364/oe.23.006058Jensen, J. B., Hoiby, P. E., Emiliyanov, G., Bang, O., Pedersen, L. H., & Bjarklev, A. (2005). Selective detection of antibodies in microstructured polymer optical fibers. Optics Express, 13(15), 5883. doi:10.1364/opex.13.005883Emiliyanov, G., Høiby, P., Pedersen, L., & Bang, O. (2013). Selective Serial Multi-Antibody Biosensing with TOPAS Microstructured Polymer Optical Fibers. Sensors, 13(3), 3242-3251. doi:10.3390/s130303242Hassan, H. U., Janting, J., Aasmul, S., & Bang, O. (2016). Polymer Optical Fiber Compound Parabolic Concentrator fiber tip based glucose sensor: in-Vitro Testing. IEEE Sensors Journal, 1-1. doi:10.1109/jsen.2016.2606580Yuan, W., Khan, L., Webb, D. J., Kalli, K., Rasmussen, H. K., Stefani, A., & Bang, O. (2011). Humidity insensitive TOPAS polymer fiber Bragg grating sensor. Optics Express, 19(20), 19731. doi:10.1364/oe.19.019731Johnson, I. P., Yuan, W., Stefani, A., Nielsen, K., Rasmussen, H. K., Khan, L., … Bang, O. (2011). Optical fibre Bragg grating recorded in TOPAS cyclic olefin copolymer. Electronics Letters, 47(4), 271. doi:10.1049/el.2010.7347Markos, C., Stefani, A., Nielsen, K., Rasmussen, H. K., Yuan, W., & Bang, O. (2013). High-T_g TOPAS microstructured polymer optical fiber for fiber Bragg grating strain sensing at 110 degrees. Optics Express, 21(4), 4758. doi:10.1364/oe.21.004758Woyessa, G., Fasano, A., Stefani, A., Markos, C., Nielsen, K., Rasmussen, H. K., & Bang, O. (2016). Single mode step-index polymer optical fiber for humidity insensitive high temperature fiber Bragg grating sensors. Optics Express, 24(2), 1253. doi:10.1364/oe.24.001253Woyessa, G., Fasano, A., Markos, C., Stefani, A., Rasmussen, H. K., & Bang, O. (2016). Zeonex microstructured polymer optical fiber: fabrication friendly fibers for high temperature and humidity insensitive Bragg grating sensing. Optical Materials Express, 7(1), 286. doi:10.1364/ome.7.000286Stefani, A., Nielsen, K., Rasmussen, H. K., & Bang, O. (2012). Cleaving of TOPAS and PMMA microstructured polymer optical fibers: Core-shift and statistical quality optimization. Optics Communications, 285(7), 1825-1833. doi:10.1016/j.optcom.2011.12.033Nielsen, K., Rasmussen, H. K., Adam, A. J., Planken, P. C., Bang, O., & Jepsen, P. U. (2009). Bendable, low-loss Topas fibers for the terahertz frequency range. Optics Express, 17(10), 8592. doi:10.1364/oe.17.008592Nielsen, K., Rasmussen, H. K., Jepsen, P. U., & Bang, O. (2010). Broadband terahertz fiber directional coupler. Optics Letters, 35(17), 2879. doi:10.1364/ol.35.002879Anthony, J., Leonhardt, R., Argyros, A., & Large, M. C. J. (2011). Characterization of a microstructured Zeonex terahertz fiber. Journal of the Optical Society of America B, 28(5), 1013. doi:10.1364/josab.28.001013Woyessa, G., Fasano, A., Markos, C., Rasmussen, H. K., & Bang, O. (2017). Low Loss Polycarbonate Polymer Optical Fiber for High Temperature FBG Humidity Sensing. IEEE Photonics Technology Letters, 29(7), 575-578. doi:10.1109/lpt.2017.2668524Johnson, I. P., Kalli, K., & Webb, D. J. (2010). 827 nm Bragg grating sensor in multimode microstructured polymer optical fibre. Electronics Letters, 46(17), 1217. doi:10.1049/el.2010.1595Stefani, A., Wu Yuan, Markos, C., & Bang, O. (2011). Narrow Bandwidth 850-nm Fiber Bragg Gratings in Few-Mode Polymer Optical Fibers. IEEE Photonics Technology Letters, 23(10), 660-662. doi:10.1109/lpt.2011.2125786Hu, X., Pun, C.-F. J., Tam, H.-Y., Mégret, P., & Caucheteur, C. (2014). Highly reflective Bragg gratings in slightly etched step-index polymer optical fiber. Optics Express, 22(15), 18807. doi:10.1364/oe.22.018807Hu, X., Pun, C.-F. J., Tam, H.-Y., Mégret, P., & Caucheteur, C. (2014). Tilted Bragg gratings in step-index polymer optical fiber. Optics Letters, 39(24), 6835. doi:10.1364/ol.39.006835Sáez-Rodríguez, D., Nielsen, K., Rasmussen, H. K., Bang, O., & Webb, D. J. (2013). Highly photosensitive polymethyl methacrylate microstructured polymer optical fiber with doped core. Optics Letters, 38(19), 3769. doi:10.1364/ol.38.003769Hu, X., Woyessa, G., Kinet, D., Janting, J., Nielsen, K., Bang, O., & Caucheteur, C. (2017). BDK-doped core microstructured PMMA optical fiber for effective Bragg grating photo-inscription. Optics Letters, 42(11), 2209. doi:10.1364/ol.42.002209Statkiewicz-Barabach, G., Kowal, D., Mergo, P., & Urbanczyk, W. (2015). Comparison of growth dynamics and temporal stability of Bragg gratings written in polymer fibers of different types. Journal of Optics, 17(8), 085606. doi:10.1088/2040-8978/17/8/085606Marques, C., Pospori, A., Demirci, G., Çetinkaya, O., Gawdzik, B., Antunes, P., … Webb, D. (2017). Fast Bragg Grating Inscription in PMMA Polymer Optical Fibres: Impact of Thermal Pre-Treatment of Preforms. Sensors, 17(4), 891. doi:10.3390/s17040891Bundalo, I.-L., Nielsen, K., Markos, C., & Bang, O. (2014). Bragg grating writing in PMMA microstructured polymer optical fibers in less than 7 minutes. Optics Express, 22(5), 5270. doi:10.1364/oe.22.005270Oliveira, R., Bilro, L., & Nogueira, R. (2015). Bragg gratings in a few mode microstructured polymer optical fiber in less than 30 seconds. Optics Express, 23(8), 10181. doi:10.1364/oe.23.010181Lacraz, A., Polis, M., Theodosiou, A., Koutsides, C., & Kalli, K. (2015). Femtosecond Laser Inscribed Bragg Gratings in Low Loss CYTOP Polymer Optical Fiber. IEEE Photonics Technology Letters, 27(7), 693-696. doi:10.1109/lpt.2014.2386692Theodosiou, A., Lacraz, A., Stassis, A., Koutsides, C., Komodromos, M., & Kalli, K. (2017). Plane-by-Plane Femtosecond Laser Inscription Method for Single-Peak Bragg Gratings in Multimode CYTOP Polymer Optical Fiber. Journal of Lightwave Technology, 35(24), 5404-5410. doi:10.1109/jlt.2017.2776862Yuan, W., Stefani, A., Bache, M., Jacobsen, T., Rose, B., Herholdt-Rasmussen, N., … Bang, O. (2011). Improved thermal and strain performance of annealed polymer optical fiber Bragg gratings. Optics Communications, 284(1), 176-182. doi:10.1016/j.optcom.2010.08.069Bundalo, I.-L., Nielsen, K., Woyessa, G., & Bang, O. (2017). Long-term strain response of polymer optical fiber FBG sensors. Optical Materials Express, 7(3), 967. doi:10.1364/ome.7.00096

Monoclonal T-Cell Receptors: New Reagents for Cancer Therapy

Adoptive transfer of antigen-specific T lymphocytes is an effective form of immunotherapy for persistent virus infections and cancer. A major limitation of adoptive therapy is the inability to isolate antigen-specific T lymphocytes reproducibly. The demonstration that cloned T-cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. TCR gene-modified lymphocytes display antigen-specific function in vitro, and were shown to protect against virus infection and tumor growth in animal models. A recent trial in humans demonstrated that TCR gene-modified T cells persisted in all and reduced melanoma burden in 2/15 patients. In future trials, it may be possible to use TCR gene transfer to equip helper and cytotoxic T cells with new antigen-specificity, allowing both T-cell subsets to cooperate in achieving improved clinical responses. Sequence modifications of TCR genes are being explored to enhance TCR surface expression, while minimizing the risk of pairing between introduced and endogenous TCR chains. Current T-cell transduction protocols that trigger T-cell differentiation need to be modified to generate “undifferentiated” T cells, which, upon adoptive transfer, display improved in vivo expansion and survival. Both, expression of only the introduced TCR chains and the production of naïve T cells may be possible in the future by TCR gene transfer into stem cells

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

Heterogeneous run-and-tumble motion accounts for transient non-Gaussian super-diffusion in haematopoietic multi-potent progenitor cells

Multi-potent progenitor (MPP) cells act as a key intermediary step between haematopoietic stem cells and the entirety of the mature blood cell system. Their eventual fate determination is thought to be achieved through migration in and out of spatially distinct niches. Here we first analyze statistically MPP cell trajectory data obtained from a series of long time-course 3D in vivo imaging experiments on irradiated mouse calvaria, and report that MPPs display transient super-diffusion with apparent non-Gaussian displacement distributions. Second, we explain these experimental findings using a run-and-tumble model of cell motion which incorporates the observed dynamical heterogeneity of the MPPs. Third, we use our model to extrapolate the dynamics to time-periods currently inaccessible experimentally, which enables us to quantitatively estimate the time and length scales at which super-diffusion transitions to Fickian diffusion. Our work sheds light on the potential importance of motility in early haematopoietic progenitor function

Manipulating niche composition limits damage to haematopoietic stem cells during Plasmodium infection.

Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and-coupled with reactive oxygen species quenching-enables partial rescuing of HSC function.Wellcome Trust, Blood Cancer UK, CRUK, MRC, BBSRC, ERC, Royal Societ

Human MHC Class I-restricted high avidity CD4 + T cells generated by co-transfer of TCR and CD8 mediate efficient tumor rejection in vivo

In this study, we generated human MHC Class I-restricted CD4+ T cells specific for Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two herpesviridae associated with lymphoma, nasopharyngeal carcinoma and medulloblastoma, respectively. Retroviral transfer of virus-specific, HLA-A2-restricted TCR-coding genes generated CD4+ T cells that recognized HLA-A2/peptide multimers and produced cytokines when stimulated with MHC Class II-deficient cells presenting the relevant viral peptides in the context of HLA-A2. Peptide titration revealed that CD4+ T cells had a 10-fold lower avidity than CD8+ T cells expressing the same TCR. The impaired avidity of CD4+ T cells was corrected by simultaneously transferring TCR- and CD8-coding genes. The CD8 co-receptor did not alter the cytokine signature of CD4+ T cells, which remained distinct from that of CD8+ T cells. Using the xenogeneic NOD/SCID mouse model, we demonstrated that human CD4+ T cells expressing a specific TCR and CD8 can confer efficient protection against the growth of tumors expressing the EBV or CMV antigens recognized by the TCR. In summary, we describe a robust approach for generating therapeutic CD4+ T cells capable of providing MHC Class I-restricted immunity against MHC Class II-negative tumors in vivo