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Preparatory Studies to Introduce Regulatory T Cells in Clinical Transplantation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Transplantation Surgery.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Solid organ transplantation has evolved from being an experimental procedure to a life-saving treatment for patients with end-stage organ failure. The risk of losing a transplant due to acute rejection is very low with the use of modern immunosuppressive protocols and the short-term results are impressive. However, long-term outcomes are suboptimal and transplant recipients are at increased risks for severe complications such as cancers, opportunistic infections and cardiovascular events. The previous struggle to achieve short-term survival has turned into a search for new strategies to improve patient and transplant longevity.

Regulatory T cells (TRegs), a subset of T cells, occur naturally in the immune system and have the capacity to down regulate immune responses. Under normal conditions they maintain self-tolerance and prevent excessive immune activation. Functional TReg defects lead to a massive autoimmune response and are not compatible with life. Preclinical data support that TRegs can be used as a cell therapy to prevent transplant rejection, with the potential to minimize the need for traditional immunosuppression and improve the long-term outcome.

This thesis aims to enhance the translation of TReg cell therapy to clinical organ transplantation. In particular, strategies for isolation and expansion of TRegs from uremic patients awaiting kidney transplantation have been assessed. A non-invasive imaging technique to study T cell products after intravenous administration was developed, for use in future clinical trials. The performance of a novel cell purification technique was investigated to potentially improve the clinical production of TRegs.

The thesis demonstrates that TRegs can be isolated and expanded from uremic patients to display potent suppressive properties in vitro. The mode of isolation and expansion affect the functional characteristics, where cells purified with cytometry based techniques and expanded with mature dendritic cells were the most advantageous. T cells can be labeled using the radioactive tracer [111In]oxine with preserved viability and subsequently followed in vivo with SPECT/CT for more than 1 week after intravenous administration. The use of microfluidic switch technology offers a novel way of purifying TRegs at high speed, purity and viability, under conditions compatible with clinical use.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 80 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 983
Keyword [en]
Transplantation, Tolerance, Cell therapy, Regulatory T cell, In vivo imaging, Cell purification
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:uu:diva-220873ISBN: 978-91-554-8907-6 (print)OAI: oai:DiVA.org:uu-220873DiVA: diva2:706830
Public defence
2014-05-10, Sal X, Universitetshuset, Biskopsgatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-04-15 Created: 2014-03-21 Last updated: 2014-04-29
List of papers
1. Isolation, expansion and functional assessment of CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation
Open this publication in new window or tab >>Isolation, expansion and functional assessment of CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation
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2012 (English)In: Transplant Immunology, ISSN 0966-3274, E-ISSN 1878-5492, Vol. 26, no 1, 27-33 p.Article in journal (Refereed) Published
Abstract [en]

Background: The immunosuppressive properties of regulatory T cells have emerged as an attractive tool for the development of immunotherapies in various disease contexts, e.g. to treat transplantation induced immune reactions. This paper focuses on the process of obtaining and functionally characterizing CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation.

Methods: From October 2010 to March 2011 uremic patients awaiting living donor kidney transplantation, and their corresponding kidney donors, were enrolled in the study. A total of seven pairs were included. Isolation of CD4+CD25+FoxP3+ regulatory T cells was performed by magnetic activated cell sorting of peripheral blood mononuclear cells obtained from the uremic patients. Donor specific Tr1 cells were differentiated by repetitive stimulation of immature CD4+ T cells with immature dendritic cells, with the T cells coming from the future kidney recipients and the dendritic cells from the corresponding kidney donors. Cells were then expanded and functionally characterized by the one-way mixed leukocyte reaction and assessment of IL-10 production. Phenotypic analysis was performed by flow cytometry.

Results: The fraction of CD4+CD25+FoxP3+ regulatory T cells after expansion varied from 39.1 to 50.4% and the cells retained their ability to substantially suppress the mixed leukocyte reaction in all but one patient (3.8–19.2% of the baseline stimulated leukocyte activity, p<0.05). Tr1 cells were successfully differentiated from all but one patient and produced high levels of IL-10 when stimulated with immature dendritic cells (1,275–11,038% of the baseline IL-10 secretion, pb0.05).

Conclusion: It is practically feasible to obtain and subsequently expand CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients without loss of function as assessed by in vitro analyses. This forms a base for adoptive regulatory T cell therapy in the setting of living donor kidney transplantation.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-170517 (URN)10.1016/j.trim.2011.09.003 (DOI)000300203800004 ()
Available from: 2012-03-13 Created: 2012-03-12 Last updated: 2017-12-07Bibliographically approved
2. Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials
Open this publication in new window or tab >>Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials
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2013 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 173, no 2, 310-322 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Surgery
Identifiers
urn:nbn:se:uu:diva-199952 (URN)10.1111/cei.12112 (DOI)000321287500016 ()23607776 (PubMedID)
Available from: 2013-05-17 Created: 2013-05-17 Last updated: 2017-12-06Bibliographically approved
3. Imaging the in vivo fate of human T cells following transplantation in immunoincompetent mice - Implications for clinical cell therapy trials
Open this publication in new window or tab >>Imaging the in vivo fate of human T cells following transplantation in immunoincompetent mice - Implications for clinical cell therapy trials
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2013 (English)In: Transplant Immunology, ISSN 0966-3274, E-ISSN 1878-5492, Vol. 29, no 1-4, 105-108 p.Article in journal (Refereed) Published
Abstract [en]

Many forms of adoptive T cell therapy are on the verge of being translated to the clinic. To gain further insight in their immunomodulating functions and to optimize future clinical trials it is essential to develop techniques to study their homing capacity. CD4+ T cells were labeled using [In-111]oxine, and the radioactive uptake was determined in vitro before intravenous injection in immunodeficient mice. In vivo biodistribution of [In-111] oxine-labeled cells or tracer alone was subsequently measured by mu SPECT/CT and organ distribution. CD4+ T cells incorporated [In-111]oxine with higher labeling yield using Ringer-Acetate compared to 0.9% NaCl. Cellular viability after labeling with [In-111]oxine was not compromised using less than 0.4 MBq/million cells. After intravenous infusion CD4+ T cells preferentially homed to the liver (p < 0.01) and spleen (p < 0.05). This study presents a protocol for labeling of T cells by [In-111]oxine with preserved viability and in vivo tracking by SPECT for up to 8 days, which can easily be translated to clinical cell therapy trials. 

Keyword
T cell, In vivo imaging, Cell therapy, SPECT/CT
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-216763 (URN)10.1016/j.trim.2013.09.009 (DOI)000329145600018 ()
Available from: 2014-01-24 Created: 2014-01-24 Last updated: 2017-12-06Bibliographically approved
4. Purification of regulatory T cells with the use of a fully enclosed high-speed microfluidic system
Open this publication in new window or tab >>Purification of regulatory T cells with the use of a fully enclosed high-speed microfluidic system
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2014 (English)In: Cytotherapy, ISSN 1465-3249, E-ISSN 1477-2566, Vol. 16, no 10, 1384-1389 p.Article in journal (Refereed) Published
Abstract [en]

Background aims. Despite promising advances in cellular therapies, it will be difficult to fully test or implement new therapies until advances are made in the processes for cell preparation. This study describes the use of an advanced prototype of a flow-cytometry cell purification system constructed for operation in a clinical environment to prepare regulatory T cells defined as CD4(+)/CD25(bright)/CD127(neg/low). Methods. The sort performance of the Gigasort system was directly compared with available droplet sorters using mixtures of highly fluorescent and non-fluorescent 5-mu m polystyrene particles. CD4(+)-enriched cell preparations were processed with the use of a sterile, disposable fluid handling unit with a chip containing parallel microfluidic-based sorters. Results. Similar purity and sort efficiency as found with droplet sorters were obtained with the 24-channel chip sorter system. Starting with 450 million fresh peripheral blood mononuclear cells, 150,000 to 1.7 million cells that were, on average, 85% FoxP3-positive and 97% viable, were obtained in <4 h. Conclusions. This study presents a technology adapted to regulatory requirements for clinical cell purification and that achieves high throughput and cell-friendly conditions by use of a microfluidic chip with 24 parallel microsorters, providing a rapid, sterile method of purifying regulatory T cells accurately and with excellent viability.

National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-220872 (URN)10.1016/j.jcyt.2014.05.016 (DOI)000342396800007 ()
Available from: 2014-03-21 Created: 2014-03-21 Last updated: 2017-12-05Bibliographically approved

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