Division of Cellular Immunology and Immunohematology Head

DIVISION OF CELLULAR IMMUNOLOGY AND IMMUNOHEMATOLOGY
Division of Cellular Immunology and Immunohematology
Head: Winfried F. Pickl, MD, Associate Professor
Current Members
Victoria M. Reichl-Leb, PostDoc
Klaus G. Schmetterer, PostDoc
Daniela Wojta-Stremayr, PhD Student
Alina Neunkirchner, PhD Student
Ulrike Körmöczi, Biomed. Scientist
Arno Rottal, Biomed. Scientist
Doris Trapin, Scientist
Past Members
Hans J. Kueng, PhD Student
Karina Schuch, Biomed. Scientist
Jakob Glaser, BSc Student
Lukas Mager, PhD Student
Antigen presentation by professional antigen-presenting cells (APC) is considered to be the
first step towards the initiation of an adaptive immune response by naïve T-cells. To that end,
antigens are presented as peptide/major histocompatibility complexes (pMHC) on APC to the
antigen receptor of T lymphocytes. For sustained T-cell activation to occur, a number of
additional molecules expressed on the surface of APC have to synergize with pMHC to
stimulate a given antigen-specific T-cell. In addition, soluble factors in the form of cytokines
critically contribute to the special milieu during T-cell activation. APC 'talk' to T-cells only
upon intimate physical interaction. The immunological synapse represents the cell biological
correlate of this APC-T-cell interaction. To better understand and describe the processes
within the immunological synapse we have started to molecularly rebuild the synapse
recently. For that purpose we have created novel reductionist antigen-presenting platforms
based on virus-like particles (VLP) and artificial APC. To also cover the T-cell side of the
synapse in due breadth and depth, we have embarked on the cloning, molecular
characterization and functional expression of human relevant T-cell antigen receptors (TCR)
recognizing human-relevant major allergens from birch and mugwort. Along those lines we
have created novel in vitro and in vivo models to study immunomodulation and regulation of
T-cell responses of human-relevant diseases, such as IgE-dependent allergies. Our studies
will contribute to a better understanding of the pathophysiology of allergies, autoimmune and
infectious diseases and may thus lead to novel strategies for their cure.
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Fig. 1: The TCR functions as a
molecular switch to induce Treg
function: TCR tg Treg which are
properly activated by their cognate
antigen initiate their regulatory function.
In the absence of antigen-specific
activation, they do not exert regulatory
function.
Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T-cells suppress Bet
v 1-specific T-cell effector function in an activation-dependent manner.
Naturally occurring CD4+CD25highFoxp3+ regulatory T-cells (Treg) orchestrate tolerance to
self-antigens by suppressing effector T-cell function. Consequently, protocols to induce Treg
function and elevate their numbers have gained increased attractiveness as therapeutic
options in recent years. We have previously shown that human T-cell receptor (TCR) αβchains specific for allergen-derived epitopes confer allergen specificity to peripheral blood Tcells of individuals with and without allergy. Klaus Schmetterer and colleagues (J Allergy Clin
Immunol, 2011, 127:238-45) studied the feasibility of generating allergen-specific human
Treg by retroviral transduction of a transcription unit encoding forkhead box protein 3
(FOXP3) and allergen-specific TCR αβ-chains. cDNAs encoding the α- and β-chains of a Bet
v 1142-153-specific TCR (TCR-α variable region 6/TCR-β variable region 20) and human
FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit
from an IRES-GFP-containing retroviral vector. Transduced T-cells displayed a Treg
phenotype with clear-cut up-regulation of CD25, CD39 and CTLA-4. The transduced cells
were hyporesponsive in cytokine production and, like naturally occurring Treg (nTreg), did not
proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was
inducible upon additional exposure to exogenous IL-2. In co-culture experiments,
TRAV6+TRBV20+FOXP3+ transgenic T-cells, unlike FOXP3+ single transgenic T-cells or
nTreg, significantly suppressed T-cell cytokine production and proliferation of corresponding
allergen-specific effector T-cells in an allergen-specific manner. Consequently, it could be
shown that TCRs function as a molecular switch to turn-on the regulatory program of Treg
(Fig. 1). Customized Treg cells might become useful for tolerance induction therapies in
individuals with allergic and other immune-mediated diseases. Future studies planned to be
performed in TCR tg mice equipped with human-relevant allergen-specific TCRs and MHC
molecules will clarify the Tregs’ potency in vivo. This work has been performed within the
framework of the SFB F18 ‘Molecular and immunological strategies for prevention, diagnosis
and treatment of Type 1 allergies’ granted by the Austrian Science Fund.
Human TCR transgenic Bet v 1-specific Th1 cells suppress the effector function of Bet
v 1-specific Th2 cells.
Birch pollen is one of the main causes of pollinosis in the Northern Hemisphere from spring
to early summer. More than 95% of birch pollen-allergic individuals mount an IgE antibody
response against Bet v 1, the major birch pollen allergen. T-cells from these patients
preferentially recognize the immunodominant epitope Bet v 1142-153. Moreover, birch pollenallergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop
allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological
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DIVISION OF CELLULAR IMMUNOLOGY AND IMMUNOHEMATOLOGY
cross-reactivity. In fact the major T-cell epitope Bet v 1142-153 plays an important role in crossreactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this
study, Alina Neunkirchner and colleagues (J Immunol, 2011,187:4077-87) have cloned and
functionally analyzed a human αβ TCR specific for the immunodominant epitope Bet v 1142153. cDNAs encoding TCR α- and β-chains were amplified from a Bet v 1142-153-specific T-cell
clone, introduced into Jurkat T-cells and peripheral blood T lymphocytes of allergic and
nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCR tg) Tcells responded in an allergen-specific and co-stimulation-dependent manner to APCs either
pulsed with Bet v 1142-153 peptide or co-expressing invariant chain::Bet v 1142-153 fusion
proteins. Moreover, TCR tg T-cells responded to Bet v 1-related food and tree pollen
allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1142153-presenting but not Bet v 14-15-presenting artificial APCs co-expressing membrane-bound
IL-12 polarized allergen-specific TCR tg T-cells toward a Th1 phenotype, producing high
levels of IFN-γ.
Fig. 2: Suppression of Th2
signature cytokines by Th1
polarized T cells: TCR tg T cells
have been activated with aAPC
expressing cognate antigen plus
membrane-bound IL-12. These
Th1-like cells were co-cultured
with Th2 cells and their effector
cytokines evaluated upon antigenspecific stimulation.
Of significance, co-culture of such Th1-polarized T-cells with allergen-specific Th2differentiated T-cells significantly suppressed Th2 effector cytokine production (Fig. 2). These
data suggest that human allergen-specific TCR can transfer the fine specificity of the original
T-cell clone to heterologous T-cells, which in turn can be instructed to modulate the effector
function of the disease initiating/perpetuating allergen-specific Th2-differentiated T-cells.
These studies were performed in close collaboration with Drs. Barbara Bohle and Beatrice
Jahn-Schmid, Institute of Pathophysiology and Allergy Research, Vienna, and Biomay AG in
the framework of the Christian Doppler Laboratory for Immunomodulation funded by the
Christian Doppler Society.
Significant differences in B-cell subpopulations characterize patients with chronic
graft-versus-host disease-associated dysgammaglobulinemia.
In a collaborative effort with the Department of Bone Marrow Transplantation, Drs. Hildegard
Greinix and Zoya Kuzmina, MUW, (Blood, 2011, 117:2265-74) we have extended our initial
investigations on the relationship between B-cell homeostasis and graft versus host disease
(GvHD). In fact, manifestations of chronic graft-versus-host disease (cGVHD) can resemble
those seen in immunodeficiency states and autoimmune disorders. Previous reports have
suggested an involvement of B-cells in the pathogenesis of cGVHD. Consequently, we
investigated B-lymphocyte subpopulations in cGVHD cohorts defined by serum
immunoglobulin G (IgG) levels to characterize novel biomarkers for impairment of humoral
immunity after allogeneic hematopoietic stem cell transplantation (HSCT). Seventy-six
patients were enrolled a median of 46 months after HSCT. The hypogammaglobulinemia
group had significantly diminished CD19+ B-cells (165 vs 454 vs 417 × 10⁶L) with elevated
CD19+CD21low immature (16.5%, 7.7%, and 9.1%) and CD19+CD21int-highCD38highIgMhigh
transitional (10.5% vs 4.2% vs 6.3%) B-cell proportions compared with the
normogammaglobulinemia and hypergammaglobulinemia groups. CD19+CD10-CD27CD21high naive B-cells were highly elevated in all patients with cGVHD. CD19+CD27+IgD+
non-class-switched (4 vs 12 vs 11 × 10⁶/L) and class-switched (7 vs 35 vs 42 × 10⁶/L)
memory B-cells were significantly lower in the hypogammaglobulinemia group compared with
the others. Besides significantly higher B-cell activation factor (BAFF)/B-cell ratios,
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DIVISION OF CELLULAR IMMUNOLOGY AND IMMUNOHEMATOLOGY
significantly more cGVHD patients with hypergammaglobulinemia had autoantibodies
compared with the hypogammaglobulinemia subgroup (68% vs 24%, P = .024). In
conclusion, B-cell subpopulations can serve as novel cellular biomarkers for
immunodeficiency and autoimmunity indicating different pathogenetic mechanisms of cGVHD
and encouraging future prospective longitudinal studies.
Crucial role for anchor dimensions of artificially membrane-bound cytokines applied
as ‘natural adjuvants’
‘Natural adjuvants’ in the form of cytokines co-administered along with the immunogen have
been applied in the past as a direct approach to improve immune system function. Besides
fostering humoral immunity, cytokines also have the potential to co-stimulate strong cellular
immune responses, which is imperative for the targeting of facultative intracellular pathogens
and tumor cells. The application of soluble cytokines may lead, however, to systemic adverse
reactions. This is mainly due to ‘overdosing’, which is necessary to reach pharmacologically
relevant concentrations of the agents in target tissues. We have shown recently that viruslike particles (VLP) can be decorated with functionally active, immunomodulatory molecules
of choice. Here Daniela Haiderer and colleagues report on factors that affect the potency of
cytokines to improve the immunogenicity of VLP co-expressing viral antigens. Virus-specific
T-cell responses are significantly affected by the membrane-anchors used to display the
cytokines. One (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b were
inserted between the model cytokine IL-2 and the minimal GPI-anchor acceptor sequence of
CD16b.
Fig. 3: Targeting of lipid-modified IL-2
to VLP: IL-2::2IgGPI reveals increased
targeting to VLP when compared to other
IL-2 length variants. 1 µg of IL-2::2IgGPI
VLP contained more than 15-times more
IL-2 than particles decorated with IL-2
GPI.
Lipid raft targeting was efficient for all four variants, although IL-2::1IgGPI surface expression
was impaired (Fig. 3). Targeting efficiency to VLP followed the order IL-2::2IgGPI > IL2::4IgGPI > IL-2::GPI > IL-2::1IgGPI. The biological activity of IL-2 on VLP followed the order
IL-2::2IgGPI = IL-2::GPI > IL-2::4IgGPI > IL-2::1IgGPI. When co-expressed on VLP with H2Db presenting the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP)33-41 peptide, IL2::2IgGPI was superior to IL-2::GPI to induce proliferation of primary LCMV-GP-specific P14
TCR transgenic T-cells in vitro, particularly when antigen was limited. Moreover, significantly
larger proportions of LCMV-specific T-cells proliferated in vivo upon challenge of mice with
antigen-specific VLP co-expressing IL-2::2IgGPI when compared to IL-2::GPI. Corresponding
loss-of-function variants of IL-2 (C92A) induced consistently less pronounced T-cell
proliferation. Our studies show that the optimal biological activity of membrane-anchored
cytokines decorating VLP used as convenient immunization platform cannot be predicted a
priori but has to be evaluated in vitro and in vivo with scrutiny. This work has been performed
in collaboration with Biomay AG and has been supported by a bridge grant from the Austrian
Research Promotion Agency (FFG).
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DIVISION OF CELLULAR IMMUNOLOGY AND IMMUNOHEMATOLOGY
Grants
FWF
FFG
CDG
WWTF
(Austrian Science Fund), Winfried F. Pickl, „Further development of anergosomes for
anergisation of allergen-specific T lymphocytes: The role of membrane bound cytokines
during T-cell activation’ “SFB-Molecular and immunological strategies for prevention,
diagnosis and treatment of Type I allergies” 2/2008 – 1/2012
(Austrian Research Promotion Agency) bridge project #812079, Winfried F. Pickl „Membrane
anchored growth factors as novel adjuvants for virus vaccines” 11/2006 – 11/2010
(Christian Doppler Society), Winfried F. Pickl „Molecular cloning and functional
characterization of allergen-specific TCRs“, subproject in ‘Christian Doppler Laboratory for
Immunomodulation – Module 4’ 12/2007 – 12/2014
(Wiener Wissenschafts-, Forschungs- und Technologiefonds) UIP-2010 “Ultrazentrifuge
samt Rotorset” 1/2011-2/2012
Bachelor thesis
Glaser, J.: Does signal 1 strength determine the functional output of human allergen specific T
lymphocytes? 2010 (completed).
PhD theses
Kueng, H. J., MSc.: Modulation of immune responses with virus-like particles decorated with cytokines
and/or integral membrane proteins. 2005-2010 (completed)
Schmetterer, K. G., MD.,MSc.: Modulation of allergen-specific T-cell responses using novel antigenpresenting platforms and transgenic regulatory T-cells. 2006-2011 (completed)
Neunkirchner, A., MSc.: Modulation of allergen-specific T-cell responses using in vitro and in vivo
model systems. 2006-current (ongoing)
Haiderer, D., MSc.: Development of better viral vaccines using membrane-bound lipid modified
cytokines as natural adjuvants. 2006-current (ongoing)
Awards
Clemens von Pirquet Award 2009 of the Austrian Society for Allergology and Immunology to PhDstudent Victoria Leb-Reichl for the publication: Modulation of allergen-specific T-lymphocyte
function by virus-like particles decorated with HLA class II molecules. J. Allergy Clin. Immunol.
(2009)124: 121-128; DOI:10.1016/j.jaci.2009.04.008
Sanofi-Aventis Award 2010 by Sanofi-Aventis, Austria, to PhD student Zoya Kuzmina for her
publication: Proportions of immature CD19+CD21- B lymphocytes predict the response to
extracorporeal photopheresis in patients with chronic graft-versus-host disease. Blood (2009)
114: 744-746; DOI: 10.1182/blood-2009-05-221028
Wilhelm Türk Award 2011 of the Austrian Society for Hematology and Oncology, Austria, to PhD
student Zoya Kuzmina for her publication: Significant differences in B-cell subpopulations
characterize patients with chronic graft-versus-host disease-associated dysgammaglobulinemia. Blood (2011) 117: 2265-2274; DOI 10.1182/blood-2010-07-295766
Publications 2010-2011
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Steiner, M., A. Attarbaschi, M. Dworzak, H. Strobl, W. Pickl, R. Kornmüller, O. Haas, H. Gadner, G. Mann;
On behalf of the Austrian Berlin-Frankfurt-Münster (BFM) Study Group. Cytochemically Myeloperoxidase
Positive Childhood Acute Leukemia With Lymphoblastic Morphology Treated as Lymphoblastic Leukemia.
J.Pediatr. Hematol. Oncol. 32:e4-7 (2010)
Küng, H.J., C. Manta,, D. Haiderer, V.M. Leb, K.G. Schmetterer, A. Neunkirchner, R.A. Byrne, C.
Scheinecker, P. Steinberger, B. Seed, W.F. Pickl. Fluorosomes: a convenient new reagent to detect and
block multivalent and complex receptor-ligand interactions. FASEB J. 24:1572-1582 (2010)
Gleixner, K.V., V. Ferenc, B. Peter, A. Gruze., R.A. Meyer, E. Hadzijusufovic, S. Cerny-Reiterer, M.
Mayerhofer, W.F. Pickl, C. Sillaber, P. Valent. Polo-like Kinase 1 (Plk1) as a Novel Drug Target in Chronic
Myeloid Leukemia: Overriding Imatinib Resistance with the Plk1 Inhibitor BI 2536. Cancer Res. 70:15131523 (2010)
Hadzijusufovic E., B. Peter, K.V. Gleixner, K. Schuch, W.F. Pickl, T. Thaiwong., V. Yuzbasiyan-Gurka, I.
Mirkina, M. Willmann, P. Valent. H1-receptor antagonists terfenadine and loratadine inhibit spontaneous
growth of neoplastic mast cells. Exp. Hematol.38:896-907 (2010)
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DIVISION OF CELLULAR IMMUNOLOGY AND IMMUNOHEMATOLOGY
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Lemez P., A. Attarbaschi, M.C. Béné, Y. Bertrand, G.L. Castoldi, E. Forestier, R. Garand , O.A. Haas, S.
Kagialis-Girard, W.D. Ludwig, E. Matutes, E. Mejstzíková, M.P. Pages, W. Pickl, A. Porwit, A. Orfao, R.
Schabath, J. Star , H. Strobl, P. Talmant, M.B. Van T Veer, Z. Zemanová; for the European Group for the
Immunological Characterization of Leukemias (EGIL) Childhood Near-tetraploid Acute Lymphoblastic
Leukemia: An EGIL Study on 36 Cases. Eur. J. Haematol. 85:300-308 (2010)
Böhm A., K. Sonneck, K.V. Gleixner, K. Schuch, W.F. Pickl, K. Blatt, B. Peter, H. Herrmann, G.H.
Schernthaner, H. Pehamberger, W. Rabitsch, W.R. Sperr, P. Valent. In vitro and in vivo growth-inhibitory
effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V. Exp.
Hematol. 38:744-55 (2010)
Ketteler R., V. Tomov, A. Neunkirchner, Q. Xie, W.F. Pickl, B. Seed. Host-encoded reporters for the
detection and purification of multiple enveloped viruses. J. Virol. Methods. 167:178-185 (2010)
Schmetterer K.G., M.G. Seidel, U. Körmöczi, A. Rottal, K. Schwarz, S. Matthes-Martin, P. Steinberger, W.F.
Pickl. Two Newly Diagnosed HLA Class II-Deficient Patients Identified by Rapid Vector-Based
Complementation Analysis Reveal Discoordinate Invariant Chain Expression Levels. Int. Arch. Allergy
Immunol. 152:390-400 (2010)
Leitner J., W. Kuschei, K. Grabmeier-Pfistershammer, R. Woitek, E. Kriehuber, O. Majdic, G. Zlabinger, W.F.
Pickl, P. Steinberger. T cell stimulator cells, an efficient and versatile cellular system to assess the role of
costimulator ligands in the activation of human T cells. J. Immunol. Methods 362:131-141 (2010)
Küng H.J., K-G- Schmetterer, W.F. Pickl. Lipid Rafts, Pseudotyping, and Virus-Like Particles: Relevance of a
Novel, Configurable, and Modular Antigen-Presenting Platform. Int. Arch. Allergy. Immunol. 154:89110 (2010)
Peter B., E. Hadzijusufovic, K. Blatt, K.V. Gleixner, W.F. Pickl, T. Thaiwong, V. Yuzbasiyan-Gurkan, M.
Willmann, P. Valent. KIT polymorphisms and mutations determine responses of neoplastic mast cells to
bafetinib (INNO-406). Exp. Hematol. 38:782-91 (2010)
Kuzmina Z., H.T. Greinix, R. Weigl, U. Körmöczi, A. Rottal, S. Frantal, S. Eder, W. Pickl. Significant
differences in B-cell subpopulations characterize patients with chronic graft-versus-host disease associated
dysgammaglobulinemia. Blood 117:2265-74 (2011)
Leitner, J., K. Drobits, W.F. Pickl, O. Majdic, G. Zlabinger, P. Steinberger. The effects of cyclosporine A and
azathoprine on human T cells activated by different costimulatory signals. Immunol. Lett. 140:74-80 (2011)
Kuschei, W.M., J. Leitner, O. Majdic, W.F. Pickl, G.J. Zlabinger, K. Grabmeier-Pfistershammer, P.
Steinberger. Costimulatory signals potently modulate the T cell inhibitory capaciy of the therapeutic CD11a
antibody Efalizumab. Clin. Immunol. 139:199-207 (2011)
Neunkirchner A., V.M. Leb-Reichl, K.G. Schmetterer, S. Mutschlechner, H.J. Kueng, D. Haiderer, K. Schuch,
M. Wallner, B. Jahn-Schmid, B. Bohle, W.F. Pickl. Human TCR Transgenic Bet v 1-Specific Th1 Cells
Suppress the Effector Function of Bet v 1-Specific Th2 Cells. J. Immunol. 187:4077-87 (2011)
Gleixner K.V., M. Mayerhofer, S. Cerny-Reiterer, G. Hörmann, U. Rix,K.L. Bennett, E. Hadzijusufovic, R.A.
Meyer, W.F. Pickl, J. Gotlib, H.P. Horny, A. Reiter, G. Mitterbauer-Hohendanner, G. Superti-Furga, P.
Valent. KIT-D816V-independent oncogenic signaling in neoplastic cells in systemic mastocytosis: role of
Lyn and Btk activation and disruption by dasatinib and bosutinib. Blood 118:1885-98 (2011)
Jahn-Schmid B., W.F. Pickl, B. Bohle. Interaction of allergens, major histocompatibility complex molecules,
and T cell receptors: a 'ménage à trois' that opens new avenues for therapeutic intervention in type I allergy.
Int. Arch. Allergy Immunol. 156:27-42 (2011)
Peter B., K.V. Gleixner, S. Cerny-Reiterer, H. Herrmann, V. Winter, E. Hadzijusufovic, V. Ferenc, K. Schuch,
I. Mirkina, H.P. Horny, W.F. Pickl, L. Müllauer, M. Willmann, P. Valent. Polo-like kinase-1 as a novel target
in neoplastic mast cells: demonstration of growth-inhibitory effects of small interfering RNA and the Polo-like
kinase-1 targeting drug BI 2536. Haematologica 96:672-80 (2011)
Matutes E., W.F. Pickl, M. Van't Veer, R. Morilla, J. Swansbury, H. Strobl, A. Attarbaschi, G. Hopfinger, S.
Ashley, M.C. Bene, A. Porwit, A. Orfao, P. Lemez, R. Schabath, W.D. Ludwig. Mixed-phenotype acute
leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008
classification. Blood 117:3163-71 (2011)
Schmetterer K.G., D. Haiderer, V.M. Leb-Reichl, A. Neunkirchner, B. Jahn-Schmid, H.J. Küng, K. Schuch, P.
Steinberger, B. Bohle, W.F. Pickl. Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells
suppress Bet v 1-specific T-cell effector function in an activation-dependent manner. J. Allergy Clin.
Immunol. 127:238-245 (2011)
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