Benita Wolf  

Home Institution
Medizinische Fakultät Carl-Gustav Carus, Technische Universität Dresden

Host Institution
Host Institutions:
Norris-Cotton-Cancer-Center,
Dartmouth Medical School
Research Mentors: Thomas Schwaab, MD; Marc Ernstoff, MD

E-Mail: benitawolf@gmx.de

Research Topic
see Abstract
Personal Reactions to the U.S. Experience
After my flight to Boston in August 2007, I took the Dartmouth Coach from Boston’s Logan Airport to Hanover, New Hampshire. I had not slept for over 40 hours and immediately fell asleep. Waking up, I found myself in the middle of nowhere. It was dark, no lights outside, barely cars on the interstate. All I saw were woods, mountains and the stars. After another hour, that I spent trying to find some signs of civilization and wondering where I was going, the bus finally arrived in the so called "Upper Valley". And although Dartmouth is such a remote place, I had one of the best years in my life, and this includes the time in the lab as well as private life.
I lived near Mascoma Lake in a tiny but cute apartment that I rented from one of my lab co-workers, The whole family gave me a very warm welcome. Living in the middle of the woods, directly at the lakeside, gave me the impression of instant holiday. After organizing a car, I finally got started in the lab, where I first continued the work of my BMEP predecessor, Adrian Schwarzer and started my own projects later on.
The lab is very well equipped and I got great support from everyone whenever I needed it. What impressed me most was how people interacted with each other. They are creating a friendly, positive and constructive atmosphere. Doors were always open and I did not feel this strong hierarchy that I often felt back in Germany.
Since I spent long hours in the lab, it was not easy to socialize properly. I joined a Gospel Choir and a Book Club, and also found friends among the numerous PhD students, who worked on my floor. At the beginning, I struggled sometimes with, what I thought is the American superficial affability. As I learned later, it can be seen as a kind of survival strategy in a country that unites multiple nationalities. Communicating on the least common denominator prevents people from getting too deep into any subjects that could cause arguments or offend anybody’s sensibilities.
I learned more about science and life in this year, than I would ever have expected before, and I would not want to miss this experience.
Greatest Difficulties Encountered
There were no big difficulties at all. Make sure you start organizing the DS-2019 early enough. I had great support from Dartmouth International Office and Thomas Schwaab, and it all went smoothly. I am afraid, that the greatest difficulty might actually be to get used to German circumstances again.
Most humorous incident
During my first week in Dartmouth, I received an e-mail from an administrative sender saying: "Code Red; bomb threatening; a bomb was found on floor 5, everybody has to leave the building immediately; evacuation." It did not surprise me at all, since Hillary Clinton was about to visit the hospital during the next days. By that time, I was not used to the American bias to all kinds of alarms (fire alarm, bomb alarm, etc.). I ran from my office to the lab and told everybody, that we had to leave. The others were not connected to the hospital mail distributor and would therefore not receive this kind of mail. So, everybody went home. At home, I mailed my friend Christina, who was surprisingly still in the hospital. She laughed herself to death... because I had not seen the 4 little words in the e-mail saying: "This is a drill."
Helpful Hints for Future Students
- Great resource for lab procedures, background knowledge, etc.:
- www.biochemweb.org/
- www.genome.ad.jp/kegg/kegg2.html, for path-way analysis
- Car insurance: State Farm insurance offered me a very good deal.
- AAA membership could come in handy sometimes.
- TracFone: cheap handy that has reception almost everywhere, at least in New England
- Places to go in New England: Acadia National Park (www.nps.gov/acad), White Mountains (www.visitwhitemountains.com)
- I am more than happy to help with more detailed information, just e-mail me.

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Abstract on Research Topic

Analysing immunological outcome of patients with metastatic Renal Cell Carcinoma (mRCC) undergoing immunotherapy and expression of Indoleamine 2,3-dioxygenase (IDO) in Dendritic cells (DCs) cultured under DC1 polarizing conditions

Authors:
B. Wolf, T. Schwaab, M. S. Ernstoff

Institution:
Dartmouth Hitchcock Medical Center, Lebanon, NH; Dartmouth College, Norris Cotton Cancer Center, Lebanon, NH

Introduction:
Renal cell carcinoma is the 9th leading site for cancer in the U.S., particularly for the 40% of the patients initially presenting with metastatic disease the prognosis remains poor. To date, clinical trials using cancer vaccines have been disappointing. Yet, their immunologic potential remains intriguing. Our group developed a new intranodal Dendritic cell vaccine strategy combined with high-dose Interleukin-2 and Interferon-a therapy which yielded surprisingly good results (ORR= 45%) in a group of 18 mRCC patients. Cytokines play a key role in the patients immuneresponse. Therefore we evaluated 27 cytokines, in serum of RCC patients undergoing immune therapy. Microarray analysis on whole genome of patients peripheral blood mononuclear cells was performed to characterize treatment- and response- related changes in lymphocyte gene expression.
Recent research in cancer DC based immunotherapy has focused on polarizing DCs to DC1 to enhance a TH1 immune response in vitro. Polarization of DCs occurs when cultures are stimulated with interferon-γ (IFNγ) and lipopolysaccharide (LPS). Another subset of DCs is known to express IDO and down-regulate T cell proliferation via tryptophan depletion. IDO may be expressed in an active and inactive form. We postulated that DC1 polarization conditions (IFNγ and LPS) would induce a functional form of IDO which may subsequently be deleterious to induction of T cell anti-cancer immunity. Our study compares IDO expression and function in monocytes, immature DCs, mature DCs and polarized DCs ex vivo.

Methods:
Cytokines: Pre- treatment and post- first- induction-cycle serum samples were included in the study. Serum from 8 patients (4 responders and 4 non-responders) and 5 age matched healthy donors (HD) was analysed for 27 cytokines using the Luminex® fluorescent bead technology. Student t-Test or Whitney..rank sum test was used to test for differences between groups and p<0.05 was considered significant.
Microarrays-analysis: Cell intensity files were normalized using RMA and Bioconductor. Fold change, p-Values (Student's t-test) and Rank Product were calculated to test for significant differences in gene expression. Ingenuity pathway analysis and GSEA were used for further analysis.
IDO: Peripheral blood monocytes of two healthy donors were isolated from leukapheresis product. Non-polarized DCs were cultured using either IL-4+GM-CSF+TNFα or IL4+GM-CSF+TNFα+PGE2. For polarization DCs received IL4+GM-CSF+LPS+IFNγ. Immature DCs were exposed to IL4+GM-CSF only. IDO protein expression was detected by western immunoblotting of cell lysates. IDO function was assessed using a HPLC method to measure the ratio of kynurenine (K) (the metabolic product) and tryptophan (T) in the culture media.

Results:
Cytokines: IL-1ra (IL-1 receptor antagonist), IL-4, IL-6, IL-7, IL-8, IL-12, IL-13, G-CSF, IFN-gamma, IP-10 (Inferferon gamma inducible protein), MIP-1a, MIP-1b, PDGF, RANTES, VEGF showed significant higher values in RCC patients than in HD. Treatment with IL-2 , Interferon alpha and DC vaccine resulted in significant higher serum levels of the anti-angiogenetic factor IP-10 (125.8±70.6 vs 520.6±244.5, p:0.002). Responding patients showed higher levels of IP-10 pre-treatment than non-responding patients (169±68.3 vs. 82.5±44.5, p:0.78). In responding patients, inflammatory cytokines decreased during treatment, while those cytokines showed an increase in non-responding patients. (ratio NR/R pre: 0.45±0.58 vs post: 2.02±3.60, p:0.008).
Microarray-analysis: Preliminary results show an up regulation in NK-cell signaling, B-cell signaling and IL-4 signaling in responding patients after treatment. Non-responding patients seem maintain defects in antigen presentation throughout the therapy. Significant differences were found in genetic profile of PBMCs of RCC patients compared with HD.
IDO-protein expression and function:
Western blot analysis showed a marked elevation in expression of IDO protein for DCs skewed to DC1, compared to non-polarized DCs. We detected large differences between K/T ratio of polarized DCs (range 0.6943 - 11.418 per 105 DCs) and non-polarized DCs (range 0.0009 - 0.2965 per 105 DCs). The K/T ratio for immature DCs ranged from .0006 -.0081 per 105 DCs.

Conclusion:
Cytokines and Microarray-analysis in kidney cancer patients undergoing immunotherapy:
Serum cytokine profile and genetic profile of PBMCs show differences between responding and non-responding patients before and after therapy. It might therefore be possible to evaluate biological markers that predict for response to immunotherapy.
IDO
Polarizing DCs to DC1 induces expression of functional IDO and should be considered in the evaluation of ex vivo DC maturation methods for human therapy.