Christine Krüger

Home Institution
Medizinische Hochschule Hannover

Host Institution(s)
The Johns Hopkins University, School of Medicine, Dept. of Pathology, Baltimore, MD
Mentors: Jonathan P. Schneck, M.D., Ph.D., Dr. rer.nat. Mathias Ölke

E-Mail:
 chr_krueger@gmx.de

Research Topic
see Abstract
Personal Reactions to the U.S. Experience
It was one of the best decisions of my life to go to the US. This sentence, in my humble opinion, summarizes the six months I spent in Baltimore the best. After having done one and a half years of research in Germany already, it was overwhelming for me to see some of the differences. Not only the money and the equipment of the labs, but also the whole environment at Hopkins with numerous international speakers in seminars several times a week impressed me lastingly, not to mention the Nobel reception for Peter Agre in November. When my boss saw that I was interested in and already informed about the topic I was supposed to work on and had some experience in research already, he allowed me to conduct my own experiments almost independently and gave me every support I needed before I even asked for. I am more than thankful that I had the opportunity to work in such a great lab with so many stimulating and inspiring people. However, apart from working quite some hours in the lab, I tried to get out of Baltimore as often as I could, since this is not the place where you want to spend (all) your weekends. With friends I traveled around, we saw the Atlantic and the Pacific, Niagara Falls in Canada, Austin in Texas for Christmas and a little bit in between. So, take your chance to get away, nobody will blame you for this, as long as you get some results.
Greatest Difficulties Encountered
It was quite difficult to get the J1 visa, I actually had to apply for two visa, because the US government changed the immigration laws when I just had received my first one. So please start your paperwork early, which means about 9 months in advance if you do not want to experience any bad surprises. And always be insisting, but nice to the person in charge.

Helpful Hints for Future Students

  • If you have a Deutsche Bank account, you can use the ATM of the Bank of America free of charge.
  • Try to find all the free-lunch-places, since food is quite expensive around Hopkins: The best is to attend lunch seminars almost every day, because by this you get to hear some extraordinary speakers from all different departments and good food for free in one occasion….
  • Baltimore has beautiful areas, such as Fells Point and Charles Village. The only problem: In order to get there, you have to cross several areas where you definitely do not want to be even during daytime. So please follow the rules: 1) Never go anywhere alone. 2) Call the security service, if you have to walk on campus after nightfall. They are very friendly and escort you. 3) Every place that resembles a question mark: Do not go there. 4) Get used to the sirens and the helicopter with the headlight. 5) Always have $10 in your pockets. Just in case. 6) Or get out of Baltimore on the week-ends: It is close to Washington D.C., New York and Philadelphia - and if you go to Philly on a Saturday night: visit "Comedysportz" at 2030 Sansom Street between Walnut & Chestnut and 20th & 21st. You´ll never forget it. · Do not discuss politics and religion.
  • Watch the "Tagesthemen" on the internet, unless you want to rely on CNN as your major source in news.
  • Get used to the fact that Germany equals beer, soccer and cars. And a "no!" to the war in Iraq.
  • Try to see as much of the country and the real life (!) in America as possible, maybe you get the chance to spend some days in a family. I did this for Thanksgiving, and it was an experience I do not want to miss (the Turkey was great…).

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Abstract on Research Topic

Establishment of a model for treatment of human melanoma by adoptive transfer of antigen-specific T cells in SCID/bg mice
Authors:
Christine Krüger, Mathias Ölke, Jonathan P. Schneck
Institution:
The Johns Hopkins University, School of Medicine, Department of Pathology, Baltimore, Maryland
Aim of the Study and Background:
Previous experiments have shown that human T cells cultured with artificial antigen presenting cells (aAPC) are able to lyse target cells in an antigen-specific manner. These aAPC consist of a magnetic bead, to which a stimulating anti-CD28 antibody and an HLA-A2-dimer are covalently bound. By this method, we were able to get a specificity of the T cells for the melanoma peptide Mart-1 of about 45% in four weeks with a stable expansion rate. Starting with one million cells and a precursor frequency of about 0,1%, about 25 million cells were obtained after four weeks of culture with a specificity of about 45%, which equals a total expansion rate of the peptide-specific T cells of 104 fold. The specificity of the T cells was assessed weekly by FACS (dimer- and tetramer staining).
The aim of this study was i) to assess the ability of T cells to enter a Mart-1 expressing human melanoma in vivo and ii) to explore if it is possible to prevent or delay tumor growth in an antigen-specific fashion. For this, two different human melanoma cell lines were injected in SCID/bg mice, which have a severe deficiency in B-, T- and NK cells. These mice were then treated with T cells generated on aAPC.
Materials and Methods:
CELL LINES: Human T cells specific for the melanoma antigen Mart-1 and the CMVpp65 peptide (as a negative control) were generated in vitro using bead-based artificial antigen presenting cells (aAPC). CD8+ T cells were isolated from PBMC of healthy, HLA-A2+ donors using magnetic beads and then cultured in vitro with the aAPC. To perform the in vivo experiments, human melanoma cells were subcutaneously injected into SCID/bg mice. The melanoma cell lines expressed either Mart-1 and HLA-A2 (MeI493) or only Mart-1 (MeE483) as a control.
IN VIVO STUDY: In the first set of experiments, the tumors were established for about ten days. After this, the mice were injected with human Mart-1 specific T cells from one single donor. The T cells were analyzed for expression of CD45RA, CCR7 and CD62L using flow cytometry. Additionally, the ability to lyse the tumor cells in vitro was examined in a standard 51Cr release assay. Two and five days after transfer, the tumors were explanted and analyzed for the presence of human T cells in the tumor. For this, T cells were isolated from the tumors and analyzed by a dimer- and tetramer staining for flow cytometry. In the second set of experiments, T cells and tumor cells were injected simultaneously and the mice assessed for tumor growth by measuring the tumor diameter twice a week.
Results:
The T cells displayed mainly an effector-memory phenotype (TEM) with CD45RA-, CCR7- and CD62L+ prior to injection. Besides, the T cells lysed the tumor cells in an antigen-specific manner in vitro, since only with MeI493 (Mart-1+/HLA-A2+) as a target a substantial lysis was detectable even at an E:T ratio of 1:1. Human T cells could be detected two and five days after transfer in the tumors which expressed Mart-1 in the context of HLA-A2. In contrast, in HLA-A2-/Mart-1+ tumors, only very few human T cells were detectable at these time points. When melanoma and T cells were injected simultaneously, only the mice with HLA-A2+/Mart-1+ tumors remained tumor free for a period of four weeks. All control mice developed tumors of about 15mm in diameter in the same time. Control mice were such without treatment, with HLA-A2-/Mart-1+ tumors or which were treated with T cells specific for the irrelevant CMV peptide pp65.
Discussion:
These first, preliminary results provide strong evidence that Mart-1 specific T cells from HLA-A2+ donors generated on aAPC are functioning in vivo. Thus, they detect HLA-A2+/Mart-1+ tumors in an antigen-specific manner and are able to destroy these. This effect is antigen-specific, since HLA-A2-/Mart-1+ tumors are not destroyed and T cells specific for an irrelevant antigen (CMVpp65) are not able to prevent tumor growth. Future experiments have to further define the underlying mechanisms and to further refine the experimental system.