Thymocyte-Derived BDNF Influences T-Cell Maturation at The DN3/DN4 Transition Stage

Eur. J. Immunol. 2015.
0: 113
DOI: 10.1002/eji.201444985
Cellular immune response
Thymocyte-derived BDNF influences T-cell maturation

at the DN3/DN4 transition stage
Ralf A. Linker1,2 , De-Hyung Lee1,2 , Anne-Christine Flach1 , Tanja Litke1 ,
Jens van den Brandt3 , Holger M. Reichardt3 , Thomas Lingner4 ,
Ursula Bommhardt5 , Michael Sendtner6 , Ralf Gold1,7 , Alexander Fl
ugel1
1
and Fred L
uhder
1
Department of Neuroimmunology, Institute for Multiple Sclerosis Research, The Hertie

Foundation and MPI for Experimental Medicine, University of G
ottingen Medical School,
G
ottingen, Germany
2
Department of Neurology, Friedrich-Alexander University Erlangen, Erlangen, Germany
3
Institute for Cellular and Molecular Immunology, University of G
ottingen, Medical School,
G
ottingen, Germany
4
DNA Microarray and Deep-Sequencing Facility, Department of Developmental Biochemistry,
University Medical Center G
ottingen, G
ottingen, Germany
5
Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-Guericke University,
Magdeburg, Germany
6
Institute for Clinical Neurobiology, University Hospital, University of W
urzburg, W
urzburg,
Germany
7
Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Bochum, Germany
Brain-derived neurotrophic factor (BDNF) promotes neuronal survival, regeneration, and
plasticity. Emerging evidence also indicates an essential role for BDNF outside the nervous
system, for instance in immune cells. We therefore investigated the impact of BDNF on
T cells using BDNF knockout (KO) mice and conditional KO mice lacking BDNF specifically
in this lymphoid subset. In both settings, we observed diminished T-cell cellularity in
peripheral lymphoid organs and an increase in CD4+ CD44+ memory T cells. Analysis
of thymocyte development revealed diminished total thymocyte numbers, accompanied
by a significant increase in CD4/CD8 double-negative (DN) thymocytes due to a partial
block in the transition from the DN3 to the DN4 stage. This was neither due to increased
thymocyte apoptosis nor defects in the expression of the TCR- chain or the pre-TCR. In
contrast, pERK but not pAKT levels were diminished in DN3 BDNF-deficient thymocytes.
BDNF deficiency in T cells did not result in gross deficits in peripheral acute immune
responses nor in changes of the homeostatic proliferation of peripheral T cells. Taken
together, our data reveal a critical autocrine and/or paracrine role of T-cell-derived BDNF
in thymocyte maturation involving ERK-mediated TCR signaling pathways.
Keywords: BDNF Neurotrophins T cells Thymus development
Additional supporting information may be found in the online version of this article at the
publishers web-site
Correspondence: Dr. Fred Luhder

e-mail: fred.luehder@med.uni-goettingen.de

C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Ralf A. Linker et al.
Introduction
Neurotrophins and other neurotrophic factors play an essential
role in the development and maintenance of the peripheral and
central nervous systems (CNS). They are involved in neuronal
survival, axonal growth, generation of new synaptic connections,
regulation of neuronal activity as well as synaptic and dendritic
plasticity. Moreover, they exert profound effects in a wide variety
of neuropsychiatric conditions including (de)myelination, pain,
aggression, and depression as well as drug abuse [18] and
modulate food intake [9]. Furthermore, neurotrophic factors are
essential for axonal maintenance [10] and are thought to contribute to regenerative processes after traumatic injury [1113].
These functions could be correlated with the expression and secretion of neurotrophic factors, not only in the CNS but also in
immune cells (reviewed in [14]). In particular, mRNA of all known
neurotrophins and their receptors were detected in the thymus,
spleen, and other lymphoid organs [1517], where they probably act in an autocrine and/or paracrine manner. However, the
neurotrophin action on immune cells is much less well defined.
The most widely studied neurotrophin, nerve growth factor,
has been reported to impact on thymic epithelium differentiation
and organogenesis [18], to influence the function of activated
CD4+ T cells [19] and B cells [20, 21] and to regulate cytokine
expression [22]. In addition, nerve growth factor acts on cells
of the myeloid-cell lineage by influencing microglial expression
of costimulatory and MHC class II molecules [23, 24] and by
suppressing the migration of monocytes through an activated CNS
endothelium [25].
Brain-derived neurotrophic factor (BDNF), another member of
the neurotrophin family, is widely expressed in the mammalian
CNS, especially in neurons, and binds with high affinity to its
receptor TrkB and with low affinity to p75NTR [1]. In addition to
its role in neurons, it was found that BDNF is expressed in cells of
the immune system such as T and B cells as well as macrophages
[17, 26] and can be detected in the CNS under inflammatory
conditions, for example, in multiple sclerosis [27]. Furthermore,
BDNF is implicated in the survival and activation of eosinophilic
granulocytes [28] and B-cell development [29]. However, data
concerning BDNF function are limited and its exact role in the
immune system is still unclear. We therefore asked whether BDNF
plays a role in T-cell lineage, in particular during thymocyte maturation, since BDNFs high-affinity receptor TrkB is expressed in
thymocytes, particularly at the early double-negative (DN) stage
[30]. Moreover, thymi of functionally TrkB-deficient mice show
structural changes consistent with massive lymphocyte death [31].
Expression of BDNF and its receptors was also reported in the
adult human thymus, both after involution and under conditions
of hyperplasia [32].
Here we show that lack of BDNF leads to reduced T-cell numbers in peripheral lymphoid organs. The search for the underlying
mechanism(s) revealed a partial block in thymocyte development
at the DN3 stage, which could be correlated with a reduced ERK
signaling pathway in this thymocyte subpopulation. Homeostasis

Eur. J. Immunol. 2015. 0: 113
and functional characteristics of peripheral T cells were, however,

found to be intact. These findings identify endogenous BDNF as
an important factor regulating differentiation processes within the
immune system and thus extend our knowledge on the pleiotropic
function of this neurotrophin outside the nervous system.
Results
Deficiency of BDNF results in reduced T-cell numbers
in peripheral lymphoid organs
To investigate the effect of BDNF on the T-cell compartment, we
started the analysis with mice carrying a ubiquitous disruption
of the BDNF gene (BDNF/ mice) in comparison with BDNFsufficient littermate controls. Since these mice die around 2 weeks
after birth due to severe neurological deficits, analyses were performed in early postnatal mice of 12 to 14 days of age. FACS analyses revealed a significantly reduced T-cell count in BDNF/ mice
in the spleen (Fig. 1A) and an increased CD4/CD8 ratio (Fig. 1B).
Expression of the activation/memory marker CD62L was not significantly different from control mice (data not shown), whereas
the percentage of CD4+ CD25+ cells was significantly increased
(Fig. 1C). Further analysis revealed that these cells expressed
FoxP3, identifying them as Treg cells (data not shown). To investigate whether T-cell-intrinsic BDNF is responsible for the observed
effect and to corroborate the results in a more mature immune system, we analyzed 6- to 8-week-old lckCre BDNFfl/fl mice lacking
BDNF specifically in T cells. These mice are viable, fertile and do
not display any spontaneous phenotype. Also in lckCre BDNFfl/fl
mice, we observed significantly reduced numbers of T cells in the
spleen (Fig. 1D) and lymph nodes (data not shown) and a small,
but significant, increase in the CD4/CD8 ratio of mature T cells
(Fig. 1E). Concerning activation/memory markers, CD62L expression again remained unchanged (Fig. 1G), whereas the percentage of CD4+ CD69+ (Fig. 1H) and CD4+ CD44+ T cells (Fig. 1I)
was significantly increased, pointing to a more activated/memory
phenotype of CD4+ T cells in the periphery. There was a tendency
toward increased numbers of CD4+ CD25+ cells without reaching
statistical significance (Fig. 1F).
Ubiquitous deletion of BDNF leads to a block in

thymocyte development at the DN3 stage
Reduced T-cell numbers in the periphery can be the result of
disturbed T-cell selection in the thymus or impaired peripheral
homeostasis. To test for the first possibility we investigated the
functional role of endogenous BDNF during thymocyte development in 12- to 14-day-old BDNF/ mice. At this age, the
mutant mice are already retarded in growth and have reduced
weight, but the relative proportion of the thymus to the total
body weight is unchanged as it is the case for other organs such
as liver (Supporting Information Fig. 1), ruling out unspecific
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Eur. J. Immunol. 2015. 0: 113
Figure 1. T lymphopenia in BDNF/ and lckCre BDNFfl/fl mice in the peripheral lymphoid organs. (AI) T cells were analyzed in the peripheral
lymphoid organs of (AC) 2-week-old BDNF/- and BDNF+ control mice as well as of (DI) 6- to 8-week-old lckCre BDNFfl/fl and BDNFfl/fl control
mice. (A and D) T-cell cellularity in the spleen; (B and E) CD4/CD8 ratio; and the percentage of (C and F) CD4+ CD25+ cells, (G) CD4+ CD62Lhigh ,
(H) CD4+ CD69+ , and (I) CD4+ CD44+ T cells is indicated. Data are pooled from at least two independent experiments. Each symbol represents
the value of a single mouse. n = 815 for (AC) and n = 58 for (DI). Statistical analysis was performed using the MannWhitney U test with
***p < 0.001, **p < 0.01, and *p < 0.05.
effects reflecting a wasting syndrome. Thymocyte numbers were

dramatically reduced in BDNF/ mice in comparison to littermate controls (BDNF+/+ or +/ mice, for which no difference
could be detected; Fig. 2A). Further studies showed a trend, but
no significant decrease in the percentage of mature CD4 or CD8
single-positive (SP) cells in BDNF/- mice (data not shown). In
contrast, the percentage of CD4/CD8 DN thymocytes was significantly increased (Fig. 2B and G) indicating a block in early thymocyte maturation. These findings were also reflected by the absolute
numbers of thymocyte subpopulations: the ratio of absolute cell
numbers between BDNF+ mice and BDNF/ littermates was the
lowest at the DN stage compared with the SP stage demonstrating
a relative accumulation of this subpopulation in BDNF/ mice

(Table 1). The observed developmental block at the DN stage

was further confined to the CD25+ CD44 DN3 stage (Fig. 2C
and G). The ratio of absolute numbers of DN1 to DN3 thymocytes from BDNF+ and BDNF/ mice was almost constant but
increased at the DN4 stage reflecting a relative loss of this subpopulation in BDNF/ mice (Table 1). Further analysis showed
that DN3 thymocytes from BDNF/ mice were smaller in size
(Fig. 2D) and expressed less CD27 (Fig. 2E), confirming a less
pronounced activation and differentiation status of this thymocyte
subpopulation. Detailed analysis of the DN thymocyte subpopulation for cells expressing CD3, the TCR, the TCR, and the
NK1.1 marker excluded the possibility that an aberrant accumulation of these subpopulations is responsible for the observed effect
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Figure 2. Defective thymocyte development

in BDNF/ mice. (AF) Thymocytes from 12to 14-day-old BDNF/ in comparison to
BDNF+/+ or BDNF+/ mice were analyzed by
FACS analysis. (A) Thymus cellularity, (B) the
percentage of CD4/CD8 DN thymocytes and (C)
of DN3-stage CD25+ CD44 thymocytes, (D) the
cell size of the DN3-stage thymocytes as percentage of the mean of WT mice in each individual experiment, (E) the percentage of DN3stage CD27+ thymocytes and (F) SP CD25+ CD4+
thymocytes are indicated. Data are pooled from
at least two independent experiments, with
n = 78 BDNF/ and n = 611 BDNF+ mice
with the exception of (D) with n = 18 mice
for both genotypes. Each animal is represented
by a single symbol. Statistical analysis was
performed using the MannWhitney U test
with ***p < 0.001, **p < 0.01, and *p < 0.05.
(G) Representative FACS plots of CD4/CD8 stainings (upper panel) and CD25/CD44 stainings
of CD4/CD8 DN thymocytes (lower panel) of
BDNF/ (right) and control BDNF+ mice (left).
The percentages on the respective cell populations are indicated in the quadrants. The
quadrants on the right represent a schematic
overview about the thymocyte subpopulations.
Plots are representative of at least five independent experiments performed.
(Supporting Information Fig. 2). Furthermore, the percentage of

CD4+ CD25+ SP cells representing selected SP thymocytes as well
as naturally occurring regulatory T cells (Treg) was not different
between BDNF/ and control mice (Fig. 2F). Expression of the
activation marker CD69 was similar in double-positive (DP) and
SP thymocytes in mice from all genotypes (Supporting Information Fig. 3), whereas the ratio of SP CD4 to DP thymocytes was
reduced in BDNF/ mice (data not shown). This finding together

with the reduced percentage of SP thymocytes as well as T-cell
numbers in the periphery points to a disturbed maturation from
the DP to the SP stage. Taken together, complete absence of BDNF
in the thymus results in disturbed T-cell development characterized by massively reduced thymic cellularity and a partial DN3
block.
Table 1. Absolute numbers of thymocyte subpopulations in BDNF/ mice in comparison to controlsa)
BDNF+
BDNF/
Statistical evaluation
Ratio BDNF+ / BDNF/
a)
CD4+
CD8+
DP
DN
DN1
DN2
DN3
DN4
12.98 6.2
3.12 1.6
***
4.1
8.05 6.9
1.83 1.6
**
4.4
96.9 46.4
31.3 19.9
***
3.1
8.63 6.4
2.71 2.4
***
3.1
0.88 0.6
0.32 0.26
**
2.75
0.61 0.56
0.25 0.24
*
2.4
3.1 2.3
1.28 1.14
**
2.4
3.93 3.17
0.82 0.81
***
4.8
Absolute numbers ( 106 ) of CD4 and CD8 SP, CD4/CD8 DP, and CD4/CD8 DN thymocytes as well as subpopulations from the latter are indicated:
DN1 (CD44+ CD25 ), DN2 (CD44+ CD25+ ), DN3 (CD44 CD25+ ), and DN4 (CD44 CD25 ) from BDNF-sufficient (BDNF+ , n = 16) and BDNF-deficient
12- to 14-day-old mice (BDNF/ , n = 11). *p < 0.05, **p < 0.01, ***p < 0.001.

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Figure 3. Defective thymocyte development in lckCre BDNFfl/fl mice. (AF) Thymocytes from 6- to 8-week-old lckCre BDNFfl/fl mice were analyzed
in comparison to BDNFfl/fl control mice by FACS analysis concerning (A) cellularity of the thymus; (B) the percentage of CD4+ , (C) CD8+ SP, and
(D) CD4/CD8 DN thymocytes; and the percentage of (E) DN3-stage CD25+ CD44 DN cells of total DN thymocytes and (F) CD25+ CD4+ SP thymocytes.
Each animal is represented by a single symbol, with n = 48 mice per group. Data are pooled from at least two independent experiments performed.
Statistical analysis was performed using the MannWhitney U test with ***p < 0.001, **p < 0.01, and *p < 0.05. (G) FACS plots for CD4/CD8 stainings
(upper panel) and CD25/CD44 stainings of CD4/CD8 DN thymocytes (lower panel) of lckCre BDNFfl/fl (right) and control mice (left). The percentages
on the respective cell populations are indicated in the quadrants. Plots are representative of four independent experiments performed.
T-cell-specific BDNF deficiency recapitulates the

phenotype of BDNF/ mice
Since BDNF is expressed in hematopoietic cells of different lineages [17], we investigated which cell type is responsible for the
production of functionally relevant BDNF in the thymus. To this
end, we used conditional KO mice in which BDNF is deleted in
immune cells of different lineages [33]. No differences in thymocyte subpopulations could be observed in mice lacking BDNF
specifically in cells of the myeloid lineage (LysMCre BDNFfl/fl ,
data not shown), pointing to the fact that myeloid cells do not
seem to play a major role for BDNF production in the thymus.
lckCre BDNFfl/fl mice lack BDNF selectively in thymocytes starting
from the DN2 stage and in mature T cells. Thymocyte cellularity in 6- to 8-week-old adult lckCre BDNFfl/fl mice was significantly reduced in comparison to control mice carrying two floxed
BDNF alleles but lacking the lckCre transgene (BDNFfl/fl , Fig. 3A).
The decrease in the percentage of mature CD4 SP (Fig. 3B and
G) and CD8 SP cells (Fig. 3C and G) in lckCre BDNFfl/fl mice

was even more pronounced compared with the situation in conventional BDNF/ mice. The percentage of DN thymocytes was
increased twofold (Fig. 3D and G), corroborating the results from
conventional BDNF/ mice. The partial developmental block previously seen at the DN3 stage in BDNF/ mice (Fig. 3E and
G) and the lack of any effect on the percentage of CD4+ CD25+
thymocytes (Fig. 3F) could also be confirmed in lckCre BDNFfl/fl
mice. The absolute cell numbers of thymocyte subpopulations also
reflected the data obtained with BDNF/ mice, revealing even
increased absolute numbers of DN and DN3 thymocytes in lckCre
BDNFfl/fl mice compared with controls irrespective of the overall
reduced thymic cellularity, which is mirrored in all other subpopulations (Table 2). The thymus phenotype could also be observed in
2-week-old lckCre BDNFfl/fl mice suggesting that the effect is not
age dependent (data not shown).
Thus, selective absence of BDNF in the T-cell lineage causes a
partial DN3 block as well as a reduced efficiency in the transition of
Table 2. Absolute numbers of thymocyte subpopulations in lckCre BDNFfl/fl mice in comparison to controlsa)
BDNF+
BDNF
Statistical evaluation
Ratio BDNF+ / BDNF/
a)
CD4+
CD8+
DP
DN
DN1
DN2
DN3
DN4
16.63 6.3
8.24 3.9
*
2.01
5.83 3.2
3.02 1.2
*
1.93
126.4 43.8
84.6 30.9
ns
1.49
4.85 1.4
5.63 2.5
ns
0.8
0.6 0.2
0.31 0.21
ns
1.93
0.19 0.06
0.15 0.05
ns
1.27
2.49 0.11
3.0 1.8
ns
0.83
2.27 0.63
1.7 1.37
ns
1.33
Absolute numbers ( 106 ) of CD4 and CD8 SP, CD4/CD8 DP, and CD4/CD8 DN thymocytes as well as subpopulations from the latter are indicated:
DN1 (CD44+ CD25 ), DN2 (CD44+ CD25+ ), DN3 (CD44 CD25+ ), and DN4 (CD44 CD25 ) from 6- to 8-week-old lckCre BDNFfl/fl (BDNF ) and Cre
negative BDNFfl/fl control mice (BDNF+ , n = 8 for CD4+ , CD8+ ; DP and DN and n = 4 for DN14). *p < 0.05; ns, not significant.

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DP to mature SP T cells and therefore modulates thymocyte development, mirroring the effects observed in conventional BDNF/
mice. The fact that LysMCre BDNFfl/fl mice do not show these
effects argues for the T cells themselves being the major source of
functionally relevant BDNF in the thymus.
ERK signaling in DN3 thymocytes is reduced

in the absence of BDNF
Several mechanisms could potentially contribute to the partial
DN3 block observed for BDNF-deficient thymocytes. First, BDNF
has been described as a neuronal survival factor [34]. In view
of the massive thymocyte apoptosis of functionally deficient TrkB
mice [31], and the recently discovered role of BDNF as a survival
factor for B-cell lines [35], we analyzed the spontaneous apoptosis
of BDNF/ and control thymocytes ex vivo. There was no difference in the percentages of AnnexinV+ CD4 SP and DP thymocytes
cultured for up to 22 h (Fig. 4A and data not shown). DN subpopulations showed a slightly more heterogeneous pattern with
BDNF-deficient DN1 to DN3 thymocytes having a lower apoptosis
rate (Fig. 4CE), whereas BDNF-deficient DN4-stage thymocytes
exhibiting slightly increased apoptosis (Fig. 4F). However, overall the apoptosis rate of DN thymocytes did not significantly differ
between BDNF-deficient and control cells (Fig. 4B). Similar results
were obtained for early apoptotic cells (AnnexinV+ 7AAD , data
not shown). These data argue against the possibility that increased
induction of apoptosis underlies the massive cell loss observed in
BDNF/ mice.
Another reason for diminished thymocyte transition from the
DN3 to DN4 stage could be disturbances in molecules critically
involved in this process. We therefore analyzed the intracellular
expression levels of the TCR- chain at the DN3 stage and found
no significant differences between BDNF-deficient (from lckCre
BDNFfl/fl mice) and control cells (Fig. 5A). Moreover, the expression of the pre-TCR as assessed by immunohistological staining
was similar (Fig. 5B).
Finally, signaling via the pre-TCR at the DN3 stage could be
disturbed. The analysis of phosphorylated signaling proteins by
intracellular FACS staining revealed that there was a significant
reduction in pERK expression levels in BDNF-deficient DN3 thymocytes compared with controls (Fig. 5C and D) whereas the expression levels of pAKT were identical (Fig. 5F and G). These results
were corroborated by Western blot analysis of FACS-sorted DN3
thymocytes from 13-day-old BDNF/ and control mice. Again,
DN3 cells from BDNF/ mice showed a marked decrease in
pERK levels whereas pAKT levels were similar (Fig. 5E and H).
Of note, there was no difference in pERK expression levels in DP
and SP thymocytes between BDNF/ and control mice (Supporting Information Fig. 4). pAKT levels were very low in DP and SP
thymocytes again without any difference between the genotypes
(data not shown). The same reduction of pERK expression levels in
DN3 thymocytes was observed in 6- to 8-week-old lckCre BDNFfl/fl
mice compared with BDNFfl/fl controls (Supporting Information
Fig. 5).

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BDNF deficiency does not affect T-cell diversity,

functionality, and homeostatic proliferation
After having established that thymocyte selection is affected by
BDNF deficiency, we next aimed at investigating its effect on
clonal diversity of SP thymocytes and on the survival of peripheral T cells and/or their homeostatic proliferation. To this end,
we FACS-sorted CD4+ SP thymocytes from BDNF/ and control mice, amplified cDNA for six different TCR- families and
sequenced the resulting amplicons. There was no difference in the
yield of different clones or in the frequency of individual clones
and a striking similarity in both usage and combination of different VDJ elements, suggesting a similar level of clonal diversity
in both genotypes (Supporting Information Fig. 6). To assess the
T-cell capacity of homeostatic proliferation, we purified T cells
from either lckCre BDNFfl/fl mice or BDNFfl/fl mice, labeled them
with CFSE, and transferred them into RAG1-deficient mice. Five
days later, analysis of spleen and lymph nodes revealed similar
numbers of CD4+ and CD8+ T cells of both genotypes, which
had undergone comparable homeostatic proliferation (Supporting
Information Fig. 7). Similar results were obtained when purifying
and transferring T cells from 2-week-old BDNF/ and appropriate
control mice (data not shown).
Finally, we investigated whether the partial block in thymocyte maturation as a result of BDNF deficiency would affect the
peripheral T-cell function in lckCre BDNFfl/fl mice. First, we studied proliferation and cytokine secretion of CD4+ and CD8+ T cells
after unspecific stimulation by monoclonal anti-CD3/CD28 antibodies. CD8+ T cells from the spleen proliferated more vigorously
than CD4+ T cells, but there was no difference between the genotypes (Fig. 6A), a finding that could be confirmed using purified
T cells from the lymph nodes (Fig. 6B). The T cells produced IFN-
and IL-17 upon stimulation, the levels again not being dependent
on the expression of BDNF (Fig. 6C and D). Second, to ascertain
the result of antigen-specific immune responses, we immunized
lckCre BDNFfl/fl mice and appropriate controls with MOG35-55
(MOG is myelin oligodendrocyte glycoprotein) and analyzed T-cell
responses at day 10 after immunization [33]. Despite the observed
T-cell lymphopenia in conditional KO mice, neither T-cell proliferation nor production of Th1/Th17 cytokines upon restimulation
with MOG peptide in vitro differed between lckCre BDNFfl/fl and
control mice (Fig. 6E and F).
Discussion
Besides their essential functions for development, homeostasis,
and regeneration in the CNS, neurotrophins and neurotrophic
factors also have been previously reported to affect cells of
the immune system. Here, we demonstrate a functional role of
endogenous BDNF for T cells at the level of thymocyte development. Thymocytes from mice deficient for BDNF in all cells or
specifically in the T-cell lineage displayed a partial block in thymocyte development at the DN3DN4 transition and DPSP maturation, which presumably resulted in an overall reduction of total
and SP thymocyte numbers without affecting their clonal diversity.
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Figure 4. Analysis of spontaneous apoptosis

of thymocyte subpopulations. (AF) Thymocytes
from 12- to 14-day-old BDNF+ (WT: solid lines,
n = 68) and BDNF/ mice (KO: dotted lines,
n = 68) were investigated by FACS analysis for the
presence of AnnexinV positive cells either directly
ex vivo or after 10 and 22 h of culture. The cells
were gated for (A) CD4/CD8 DP, (B) CD4/CD8 DN thymocytes, (C) CD44+ CD25 DN1, (D) CD44+ CD25+
DN2, (E) CD44 CD25+ DN3, and (F) CD44 CD25
DN4 thymocytes. The percentage of AnnexinV positive cells is shown as mean SEM, and data are
pooled from three independent experiments performed. Statistical analysis was performed using
the MannWhitney U test, only significant differences are shown with **p < 0.01.
One can assume that impaired thymocyte development is the cause

of the observed mild peripheral T lymphopenia. However, acute
adaptive immune responses were not compromised as exemplified by the unaltered T-cell proliferation and effector cytokine production both after antigen-independent anti-CD3/CD28-mediated
stimulation or MOG35-55 immunization and antigen-specific recall
responses. Furthermore, it was already previously shown that the
course of MOG-EAE was not different in lckCre BDNFfl/fl mice
compared with controls [33]. We cannot rule out, however, that
besides these obviously not compromised acute immune responses
tested here the defect in thymocyte development and the resulting
peripheral lymphopenia may influence chronic immune responses,
impair the host response against certain infections or impact
on immunological senescence. Further studies have to be performed to examine these possibilities over a longer observation
period.
BDNF has been implicated in axonal maintenance and neuronal
survival [10, 34]. It also promotes the survival of nonneuronal
cell types such as B-cell lines [35] so that massive lymphocyte
apoptosis occurs in the thymus of functionally deficient TrkB mice
[31]. Furthermore, survival signals are mandatory for thymocyte
development, as revealed in mice deficient for Akt1/Akt2, which
also display a DN3 block due to increased thymocyte apoptosis

[36]. Therefore, a similar role for BDNF in survival of developing

thymocytes is conceivable, and we first investigated the influence of BDNF deficiency on the apoptosis induction of different
thymocyte subpopulations. Surprisingly, we could not detect significantly increased overall apoptosis in BDNF/ thymocytes, suggesting that the induction of apoptosis is not the major cause for
the observed reduction of thymic cellularity. There were some differences in the apoptosis induction in the DN compartment with
DN1 to DN3 thymocytes succumbing to somewhat lower and DN4
to somewhat higher rates of apoptosis in BDNF-deficient thymocytes, but the differences were small and are probably only of, if
any, minor relevance for the observed partial DN3 block.
The main influence of BDNF deficiency on thymocyte development was seen for the transition from the DN3 to DN4 stage,
a critical step requiring the coordinate action of many different
signaling molecules. VDJ recombination at the DN3 stage leads
to the expression of the TCR- chain, which pairs with the preTCR chain conveying survival, proliferation, and transition to the
DN4 stage. These processes critically depend on the recombination machinery eventually leading to the expression of the TCR-
chain as well as components of the signaling cascade delivered
by the pre-TCR such as lck/fyn, Zap70/syk, and LAT [37]. We
therefore tested whether the lack of BDNF impaired the
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Eur. J. Immunol. 2015. 0: 113
Figure 5. pERK but not pAKT levels are reduced

in BDNF/ DN3 thymocytes. (A) Thymocytes
from lckCre BDNFfl/fl and BDNFfl/fl control mice
(n = 5) were surface-stained for CD4, CD8, CD25,
and CD44 and intracellularly for TCR- chain
expression. The cells were first plotted for CD4
against CD8, the DN population was plotted for
CD44 against CD25, and the CD44 CD25+ (DN3)
population was considered for further analysis.
Histograms referring to TCR- chain expression in
the DN3 subpopulation are shown, and the percentage of positive cells is indicated. Data are representative of three independent experiments. (B)
DN3 thymocytes from 12-day-old BDNF+/+ control
(left) and BDNF/ (right) mice were sorted, plated
on coverslips by cytospin and stained for pre-TCR
by immunohistochemistry. Bar = 20 m. Arrows
point to positive cells. (CH) Thymocytes from 12to 14-day-old BDNF/ and BDNF+/+ control mice
were stained for CD4, CD8, CD25, and CD44 and
intracellularly for pERK or pAKT. Representative
histograms of pERK (C) or pAKT (F) stainings of DN3
thymocytes of control (dotted line) and BDNF/
mice (solid line) are shown. The filled histogram
represents the control staining without primary
antibody. Quantification of pERK (D) or pAKT (G)
stainings (geometric mean SEM) is indicated.
Data shown are from n = 48 mice/group and
are pooled from at least two independent experiments. Statistical analysis was performed using
the MannWhitney U test with *p < 0.05. (E and H)
Protein lysates from sorted DN3 thymocytes were
analyzed for pERK (E) or pAKT (H) by Western blot.
Protein loading was controlled with ERK1/2 or actin
antibodies. Numbers beneath the blots indicate the
relative expression level of pERK and pAKT after
quantification and relation to protein loading controls. Blots are representative of two independent
experiments performed.
expression or function of some of those molecules. We did not

find differences in the expression levels of the TCR- chain or
the pre-TCR, but BDNF directly impacts a critical signaling pathway implicated in pre-TCR signaling. There are many signaling
pathways downstream of the high-affinity BDNF receptor TrkB
that are shared with pre-TCR signaling such as ERK and AKT
signaling [38]. We tested the phosphorylation status of AKT
and ERK and found that pERK levels were indeed reduced in
BDNF-deficient DN3 thymocytes whereas pAKT levels were not
affected. This suggests that BDNF could directly modulate thymocyte differentiation via modulating pERK levels in differentiating thymocytes. Interestingly, mice deficient in ERK1 also show a
defect in thymocyte maturation resulting in decreased SP CD4+
and CD8+ populations [39]. Diminished pERK levels were only
observed in the DN3 subpopulation but not in DP and even more

mature SP thymocytes, suggesting that BDNF deficiency influences thymocyte maturation only in a very limited window of
time and/or space. Additionally, BDNF may exert its effects on
thymocytes via activation of cAMP response element-binding protein (CREB), a transcription factor phosphorylated and activated
by pre-TCR signaling via PKC- and ERK-MAPK-mediated pathways [40]. Interestingly, CREB/ mice show a similar phenotype to BDNF/ mice including impaired development of fetal
T cells and an increase of DN thymocyte numbers but normal
T-cell development [41]. Furthermore, proliferation and IL-2
production are diminished in mice expressing a dominant-negative
form of CREB [42]. CREB is a major mediator in the BDNF signaling cascade [43]. Therefore, it is tempting to speculate that BDNF
could feed into the CREB-mediated signaling cascade required for
the development of DN thymocytes.
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Eur. J. Immunol. 2015. 0: 113
Figure 6. Peripheral T lymphopenia in lckCre

BDNFfl/fl mice does not influence T-cell function. (AD) T cells from lckCre BDNFfl/fl mice
and BDNFfl/fl littermates were purified, labeled
with CFSE, and cultured with monoclonal antiCD3/CD28 antibodies for 3 days. The percentage of proliferating CD4+ or CD8+ T cells from
the (A) spleen or (B) lymph node of lckCre
BDNFfl/fl mice and BDNFfl/fl littermate controls
is depicted. There was no proliferation in the
absence of anti-CD3/CD28 antibodies (data not
shown). In parallel experiments, the secretion of (C) IFN or (D) IL-17 from either antiCD3/CD28 stimulated or unstimulated purified
T cells from the spleen after 3 days of culture was determined. Data are presented as
mean SEM (n = 5) and are pooled from
two independent experiments. Statistical analysis was performed using the MannWhitney
U test with **p < 0.01 and *p < 0.05. (E and F)
lckCre BDNFfl/fl mice and BDNFfl/fl littermates
(control) were immunized with MOG35-55 . Ten
days later, splenocyte primary cultures were
analyzed for (E) T-cell proliferation and (F)
IFN- as well as IL-17 production in response to
MOG35-55 or polyclonal stimulation with ConA.
Data are presented as mean SEM (n = 34
mice/group) and are representative of two independent experiments. Statistical analysis was
performed using the MannWhitney U test and
did not reveal significant differences between
lckCre BDNFfl/fl and control mice.
Lack of BDNF impacted on thymocyte development and pERK

expression levels in the DN3 subpopulation irrespective of whether
it is confined to T cells or ubiquitously absent. This observation
favors the notion of T-cell-derived BDNF [26] acting in a paracrine
or autocrine fashion being responsible for the observed phenotype. Since BDNF is already expressed in DN thymocytes (data
not shown), these two possibilities cannot be distinguished by the
current approach. In contrast, BDNF from other potential sources,
such as thymic macrophages, apparently plays no or only a minor
role as evident by the lack of a thymic phenotype in LysMCre
BDNFfl/fl mice, which lack BDNF in myeloid cells. That the BDNF
potentially produced by epithelial cells plays a role in thymocyte
development could not consequently be ruled out but seems rather
unlikely. Interestingly, thymocyte subpopulations such as CD3+
DN cells ( T cells and NKT cells) or Treg cells appeared normal

or were even increased in size in the absence of BDNF whereas the

maturation of conventional thymocytes was partially impaired
at the DN3 stage, suggesting a differential dependence on BDNF
for different T-cell subsets.
One important question is the extent and the consequences
of the distortion of T-cell development by the absence of BDNF.
The partial DN3 block was evident in every setting and at every
age of the mice and, even if it was far from complete, one can
assume that it certainly impacts the thymic output with consequences for T-cell cellularity in the periphery, a compartment that
normally reacts in a very plastic manner to subtle changes in thymocyte development and output. The effect on maturation of DP
to SP thymocytes was more evident in adult lckCre BDNFfl/fl mice,
which suggests that this maturation block is able to enhance the
effect of the partial DN3 block on thymic output leading eventually
www.eji-journal.eu
10
to peripheral T lymphopenia. Whether a partially defective positive selection or enhanced negative selection is responsible for the
effect on the maturation of DP to SP thymocytes and the relative
contribution of the partial DN3 and DP maturation block to the
overall phenotype remains to be elucidated in further studies. In
summary, regarding BDNF as a factor contributing to efficient thymocyte development extends our understanding of the pleiotropic
nature of this neurotrophin beyond its well-established role in the
nervous system.
Eur. J. Immunol. 2015. 0: 113
For FACS analysis of peripheral immune cells, cells were gated

on living lymphocytes, followed by gating for CD4+ cells, and
subsequently analysis for the appropriate markers was performed.
For thymus analysis, cells were gated on living lymphocytes, followed by discrimination for CD4 and CD8. Cells negative for both
markers were analyzed for CD25 and CD44 to allow definition of
the four DN subsets. In some cases, CD25+ CD44 DN3 cells were
gated and analyzed for the appropriate markers.
CFSE labeling and cell transfer
Materials and Methods

Animal experimentation
C57Bl/6 mice were purchased from Charles River (Sulzfeld,
Germany). BDNF/ mice were generated at the Max-Planck Institute for Neurobiology, Martinsried, Germany, using D3, 129Sv
embryonic stem cells [44] and subsequently backcrossed to the
C57Bl/6 background for more than 15 generations. Conditional
BDNFfl/fl mice were backcrossed to the C57Bl/6 background for
more than ten generations [45] and were crossed with mice
expressing Cre under the control of the proximal lck promoter
[46] or the LysM promoter [47]. All control mice were BDNF+ or
BDNFfl/fl littermates. All experiments were approved by the Lower
Saxony state authorities for animal experimentation.
FACS analysis
FACS analysis was performed using a FACSCalibur or FACS Aria
II sorp from BD Biosciences (Heidelberg, Germany) and CellQuest
software as previously described [48]. The following antimouse
clones from BD were used: CD4 (H129.19 or GK 1.5), CD8a (536.7), CD3 (145 2C11), CD25 (7C4), CD44 (IM7), CD62L (Mel14),
CD69 (H1 2F3), NK1.1 (PK136), and TCR (B1). For thymus
analysis, cells were routinely plotted for CD4 against CD8, and the
CD4 CD8 population for CD44 against CD25. The CD44 CD25+
(DN3) population was subjected to further analysis. For peripheral lymphoid organs, cells were plotted for CD4 against CD8, and
the CD4+ and CD8+ populations were analyzed separately (the
gating strategy is shown in Supporting Information Fig. 8). For
intracellular staining, the FoxP3 staining kit (clone FJK-16s) from
eBioscience (San Diego, CA, USA) was used according to the manufacturers instructions. Intracellular staining for the TCR- chain
was performed using Fix-Perm (BD Biosciences) and anti-TCR
(H57-597, BD Biosciences). For the intracellular staining of phosphorylated signaling proteins, thymocytes were incubated with
anti-CD3 (clone 145-2C11, eBioscience), crossed-linked for 10 min
at 37C, fixed with Fix-Perm (BD Biosciences), and subsequently
stained for CD4, CD8, CD25, CD44 and Alexa488-conjugated
pAKT (Cell Signalling Technology, Danvers, MA, USA) or pERK
Tyr 204 (Santa Cruz Biotechnology, Dallas, TX, USA) followed
by goat-anti-rabbit APC conjugated secondary antibody (Dianova,
Hamburg, Germany).

T cells isolated from the spleen of 13-day-old BDNF/ or

BDNF+/+ littermates were purified using a pan-T-cell isolation kit
(Miltenyi, Bergisch-Gladbach, Germany) and labeled with CFSE
as previously described [49]. A total of 1 107 cells were adoptively transferred intravenously into RAG1/ mice. Five days
later, spleen and lymph node cells from the recipient mice were
analyzed by FACS staining for CD3, CD4, and CD8 expression.
Thymocyte apoptosis
Thymocytes were collected from either 13-day-old BDNF/ mice
or control littermates and cultured for up to 22 h in RPMI/10%
FCS. The apoptosis rate was determined by flow cytometry using
FITC-labeled AnnexinV (BioLegend, San Diego, CA, USA) in combination with 7AAD (Sigma, Taufkirchen, Germany) and CD4-,
CD8-, CD25-, and CD44-specific mAbs.
Cytospin and immunohistology

DN3 thymocytes were sorted using a FACS Aria II sorp (BD) from
12-day-old BDNF-deficient and control mice and plated on cover
slips using a Shandon Cytospin 4 (Thermo Scientific, Dreieich,
Germany). Cells were stained via immunocytochemistry employing the M.O.M. Kit (Vector via Linaris, Dossenheim, Germany)
with a mouse anti-pre-TCR alpha Ab (1:100; BD Bioscience) and
with anti-mouse Alexa 488 as secondary antibody (1:1000) and
antifade reagent with DAPI (all Invitrogen, Darmstadt, Germany).
Western blot
Protein extracts from FACS-sorted DN3-stage thymocytes were
prepared as described [50]. Proteins were run on a 10% SDSPAGE, transferred to nitrocellulose, and analyzed for the expression of pERK1/2 (Thr202 /Tyr204 ), pAKT (Ser473 ), total ERK1/2 (all
from Cell signalling Technology), and -actin (Santa Cruz Biotechnology). Primary antibodies were detected with mouse-anti-rabbit
antibodies (Jackson ImmunoResearch Laboratories, West Grove,
PA, USA) and the ECL detection system (Thermo Scientific Pierce,
Rockford, IL, USA). Quantification of protein expression was performed with Kodak software.
www.eji-journal.eu
Eur. J. Immunol. 2015. 0: 113
In vitro T-cell culture and ELISA

T cells from young adult lckCre BDNFfl/fl mice or BDNFfl/fl mice
as controls were purified using a negative selection purification
kit (Easy sep mouse T-cell isolation kit, Stemcell technologies,
Grenoble, France), labeled with CFSE and plated in 24-well plates
at a concentration of 2106 cells/mL and incubated with 1 g/mL
anti-CD3 (145-2C11) and 5 g/mL anti-CD28 (37.51; both from
eBioscience). After 3 days, cells were stained for CD4 and CD8 and
analyzed by FACS analysis. At the same time, the supernatant was
collected and analyzed for IFN-, and IL-17 levels by sandwich
ELISA, as previously described [51]. mAb pairs and recombinant
cytokine standards were purchased from BD for all cytokines.
20 g/mL of MOG 3555 peptide or 1.25 g/mL ConA. Proliferation was investigated by measuring 3 [H]-thymidine uptake as
described [52]. Supernatants from a parallel culture were harvested after 3 days and analyzed for IFN- and IL-17 levels by
sandwich ELISA, as described [51].
Statistical analyses
Statistics were performed after controlling for normal distribution by MannWhitney U test (PRISM software, Graph Pad, San
Diego, CA, USA). Data are given as mean SEM. The following
p-values were considered as significant: *p < 0.05, **p < 0.01,
***p < 0.001. p 0.05 was considered as not significant.
cDNA amplification and sequencing

Thymocytes from 12-day-old BDNF/ mice and WT littermates
(n = 3) were stained for CD4 and CD8 and then the CD4+ SP
thymocytes FACS-sorted using the FACS Aria II sorp. RNA was
prepared using the RNeasy Micro Kit (Qiagen, Venlo, The Netherlands) and reverse transcription was performed using the Firststrand cDNA Synthesis Kit (Thermo, Waltham, MA, USA). For the
amplification of TCR- alleles, primers specific for six different
TCR- families (V2, V3.1, V7, V8.1, V8.2, and V11) were
applied in combination with a reverse primer from the C region as
described previously [52]. PCR products from each single mouse
were pooled and sequenced using an Illumina Miseq instrument
with 2 150 bp paired-end read configuration. After basecaling
and demultiplexing individual samples using the Illumina CASAVA
pipeline, the reads were assembled with the PEAR software [53].
Unassembled and low-quality reads were disregarded from further downstream analysis. The number of assembled reads varied
between 787 351 and 2 170 962 (1 320 518 462 782) per
sample with an average read length of 179 33 bp. To identify
V/D/J rearrangements, the sample files were analyzed using the
Vidjil software [54]. For this purpose, germline data of known
V/D/J elements for Mus musculus were downloaded from the
IMGT database (www.imgt.org, as of December 2014). Analysis of
recombination yielded on average 18 333 8606 clones per sample (13 820 4601 per million reads, see Supporting Information
Fig. 6). For combined data from both genotypes, the total number
of detectable clones was 53505/56022 (control/BDNF/ ).
Acknowledgments: The authors thank M. Weig, B. Curdt, A.

Bohl, N. Meyer, and S. Seubert for excellent technical assistance; N. Kruse for help with the real-time PCR; and Cathy Ludwig for language corrections. This work was supported by the
Gemeinn
utzige Hertie-Stiftung (AZ 1.01.1/05/009 to R.L., R.G.,
and F.L.), Niedersachsen-Research Network on Neuroinfectiology
(N-RENNT) of the Ministry of Science and Culture of Lower Saxony (to A.F. and F.L.) and by grants from the Deutsche Forschungsgemeinschaft (FOR 1336 to A.F., SFB-TRR 43 to A.F. and F.L., SFB
854, TP 9 to U.B.) and the Bundesministerium f
ur Bildung und
Forschung (UNDERSTAND MS to A.F.).
Conflict of interest: The authors declare no financial or commercial conflict of interest.
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46 Hennet, T., Hagen, F. K., Tabak, L. A. and Marth, J. D., T-cell-specific deletion of a polypeptide N-acetylgalactosaminyltransferase gene by sitedirected recombination. Proc. Natl. Acad. Sci. USA 1995. 92: 1207012074.
47 Clausen, B. E., Burkhardt, C., Reith, W., Renkawitz, R. and Forster,
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48 Wust,
S., vanden Brandt, J., Tischner, D., Kleiman, A., Tuckermann,
Full correspondence: Dr. Fred Luhder,

Department of
Neuroimmunology, Institute for Multiple Sclerosis Research, The
Hertie Foundation and MPI for Experimental Medicine, University of
Gottingen,
Waldweg 33, 37073 Gottingen,
Germany
e-mail: fred.luehder@med.uni-goettingen.de
Current address: Jens van den Brandt, University of Greifswald Medical
School, Central Core & Research Facility of Laboratory Animals,
Walther-Rathenau-Str. 49a, 17489, Greifswald, Germany
J. P., Gold, R., Luhder,
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the therapeutic targets of glucocorticoids in experimental autoimmune
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49 Luhder,
F., Chambers, C., Allison, J. P., Benoist, C. and Mathis, D., Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to

Received: 2/7/2014
Revised: 18/12/2014
Accepted: 22/1/2015
Accepted article online: 27/1/2015
www.eji-journal.eu
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Thymocyte-Derived BDNF Influences T-Cell Maturation at The DN3/DN4 Transition Stage

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Thymocyte-Derived BDNF Influences T-Cell Maturation at The DN3/DN4 Transition Stage

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Eur. J. Immunol. 2015.

Cellular immune response