linker et al-2015-european journal of immunology

Eur. J. Immunol. 2015. 0: 113 Cellular immune responseDOI: 10.1002/eji.201444985 1

Thymocyte-derived BDNF influences T-cell maturationat 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 Flugel1

and Fred Luhder1

1 Department of Neuroimmunology, Institute for Multiple Sclerosis Research, The HertieFoundation and MPI for Experimental Medicine, University of Gottingen Medical School,Gottingen, Germany

2 Department of Neurology, Friedrich-Alexander University Erlangen, Erlangen, Germany3 Institute for Cellular and Molecular Immunology, University of Gottingen, Medical School,Gottingen, Germany

4 DNA Microarray and Deep-Sequencing Facility, Department of Developmental Biochemistry,University Medical Center Gottingen, Gottingen, Germany

5 Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-Guericke University,Magdeburg, Germany

6 Institute for Clinical Neurobiology, University Hospital, University of Wurzburg, Wurzburg,Germany

7 Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Bochum, Germany

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival, regeneration, andplasticity. Emerging evidence also indicates an essential role for BDNF outside the nervoussystem, for instance in immune cells. We therefore investigated the impact of BDNF onT cells using BDNF knockout (KO) mice and conditional KOmice lacking BDNF specificallyin this lymphoid subset. In both settings, we observed diminished T-cell cellularity inperipheral lymphoid organs and an increase in CD4+CD44+ memory T cells. Analysisof thymocyte development revealed diminished total thymocyte numbers, accompaniedby a significant increase in CD4/CD8 double-negative (DN) thymocytes due to a partialblock in the transition from the DN3 to the DN4 stage. This was neither due to increasedthymocyte apoptosis nor defects in the expression of the TCR- chain or the pre-TCR. Incontrast, 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 immuneresponses nor in changes of the homeostatic proliferation of peripheral T cells. Takentogether, our data reveal a critical autocrine and/or paracrine role of T-cell-derived BDNFin 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 thepublishers web-site

Correspondence: Dr. Fred Luhdere-mail: [email protected]

C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

2 Ralf A. Linker et al. Eur. J. Immunol. 2015. 0: 113

Introduction

Neurotrophins and other neurotrophic factors play an essentialrole in the development and maintenance of the peripheral andcentral nervous systems (CNS). They are involved in neuronalsurvival, axonal growth, generation of new synaptic connections,regulation of neuronal activity as well as synaptic and dendriticplasticity. Moreover, they exert profound effects in a wide varietyof neuropsychiatric conditions including (de)myelination, pain,aggression, and depression as well as drug abuse [18] andmodulate food intake [9]. Furthermore, neurotrophic factors areessential for axonal maintenance [10] and are thought to con-tribute to regenerative processes after traumatic injury [1113].These functions could be correlated with the expression and secre-tion of neurotrophic factors, not only in the CNS but also inimmune cells (reviewed in [14]). In particular, mRNA of all knownneurotrophins and their receptors were detected in the thymus,spleen, and other lymphoid organs [1517], where they proba-bly act in an autocrine and/or paracrine manner. However, theneurotrophin 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 differentiationand organogenesis [18], to influence the function of activatedCD4+ T cells [19] and B cells [20, 21] and to regulate cytokineexpression [22]. In addition, nerve growth factor acts on cellsof the myeloid-cell lineage by influencing microglial expressionof costimulatory and MHC class II molecules [23, 24] and bysuppressing the migration of monocytes through an activated CNSendothelium [25].

Brain-derived neurotrophic factor (BDNF), another member ofthe neurotrophin family, is widely expressed in the mammalianCNS, especially in neurons, and binds with high affinity to itsreceptor TrkB and with low affinity to p75NTR [1]. In addition toits role in neurons, it was found that BDNF is expressed in cells ofthe immune system such as T and B cells as well as macrophages[17, 26] and can be detected in the CNS under inflammatoryconditions, for example, in multiple sclerosis [27]. Furthermore,BDNF is implicated in the survival and activation of eosinophilicgranulocytes [28] and B-cell development [29]. However, dataconcerning BDNF function are limited and its exact role in theimmune system is still unclear. We therefore asked whether BDNFplays a role in T-cell lineage, in particular during thymocyte mat-uration, since BDNFs high-affinity receptor TrkB is expressed inthymocytes, particularly at the early double-negative (DN) stage[30]. Moreover, thymi of functionally TrkB-deficient mice showstructural changes consistent withmassive lymphocyte death [31].Expression of BDNF and its receptors was also reported in theadult human thymus, both after involution and under conditionsof hyperplasia [32].

Here we show that lack of BDNF leads to reduced T-cell num-bers in peripheral lymphoid organs. The search for the underlyingmechanism(s) revealed a partial block in thymocyte developmentat the DN3 stage, which could be correlated with a reduced ERKsignaling pathway in this thymocyte subpopulation. Homeostasis

and functional characteristics of peripheral T cells were, however,found to be intact. These findings identify endogenous BDNF asan important factor regulating differentiation processes within theimmune system and thus extend our knowledge on the pleiotropicfunction of this neurotrophin outside the nervous system.

Results

Deficiency of BDNF results in reduced T-cell numbersin peripheral lymphoid organs

To investigate the effect of BDNF on the T-cell compartment, westarted the analysis with mice carrying a ubiquitous disruptionof the BDNF gene (BDNF/ mice) in comparison with BDNF-sufficient littermate controls. Since these mice die around 2 weeksafter birth due to severe neurological deficits, analyses were per-formed in early postnatal mice of 12 to 14 days of age. FACS anal-yses revealed a significantly reduced T-cell count in BDNF/ micein the spleen (Fig. 1A) and an increased CD4/CD8 ratio (Fig. 1B).Expression of the activation/memory marker CD62L was not sig-nificantly different from control mice (data not shown), whereasthe percentage of CD4+CD25+ cells was significantly increased(Fig. 1C). Further analysis revealed that these cells expressedFoxP3, identifying them as Treg cells (data not shown). To investi-gate whether T-cell-intrinsic BDNF is responsible for the observedeffect and to corroborate the results in a more mature immune sys-tem, we analyzed 6- to 8-week-old lckCre BDNFfl/fl mice lackingBDNF specifically in T cells. These mice are viable, fertile and donot display any spontaneous phenotype. Also in lckCre BDNFfl/fl

mice, we observed significantly reduced numbers of T cells in thespleen (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 expres-sion again remained unchanged (Fig. 1G), whereas the percent-age of CD4+CD69+ (Fig. 1H) and CD4+CD44+ T cells (Fig. 1I)was significantly increased, pointing to a more activated/memoryphenotype of CD4+ T cells in the periphery. There was a tendencytoward increased numbers of CD4+CD25+ cells without reachingstatistical significance (Fig. 1F).

Ubiquitous deletion of BDNF leads to a block inthymocyte development at the DN3 stage

Reduced T-cell numbers in the periphery can be the result ofdisturbed T-cell selection in the thymus or impaired peripheralhomeostasis. To test for the first possibility we investigated thefunctional role of endogenous BDNF during thymocyte devel-opment in 12- to 14-day-old BDNF/ mice. At this age, themutant mice are already retarded in growth and have reducedweight, but the relative proportion of the thymus to the totalbody weight is unchanged as it is the case for other organs suchas liver (Supporting Information Fig. 1), ruling out unspecific


Eur. J. Immunol. 2015. 0: 113 Cellular immune response 3

Figure 1. T lymphopenia in BDNF/ and lckCre BDNFfl/fl mice in the peripheral lymphoid organs. (AI) T cells were analyzed in the peripherallymphoid 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 controlmice. (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 representsthe 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 weredramatically reduced in BDNF/ mice in comparison to litter-mate controls (BDNF+/+ or +/ mice, for which no differencecould be detected; Fig. 2A). Further studies showed a trend, butno significant decrease in the percentage of mature CD4 or CD8single-positive (SP) cells in BDNF/- mice (data not shown). Incontrast, the percentage of CD4/CD8 DN thymocytes was signifi-cantly increased (Fig. 2B and G) indicating a block in early thymo-cyte maturation. These findings were also reflected by the absolutenumbers of thymocyte subpopulations: the ratio of absolute cellnumbers between BDNF+ mice and BDNF/ littermates was thelowest at the DN stage compared with the SP stage demonstratinga relative accumulation of this subpopulation in BDNF/ mice

(Table 1). The observed developmental block at the DN stagewas further confined to the CD25+CD44 DN3 stage (Fig. 2Cand G). The ratio of absolute numbers of DN1 to DN3 thymo-cytes from BDNF+ and BDNF/ mice was almost constant butincreased at the DN4 stage reflecting a relative loss of this sub-population in BDNF/ mice (Table 1). Further analysis showedthat DN3 thymocytes from BDNF/ mice were smaller in size(Fig. 2D) and expressed less CD27 (Fig. 2E), confirming a lesspronounced activation and differentiation status of this thymocytesubpopulation. Detailed analysis of the DN thymocyte subpopula-tion for cells expressing CD3, the TCR, the TCR, and theNK1.1 marker excluded the possibility that an aberrant accumula-tion of these subpopulations is responsible for the observed effect



Figure 2. Defective thymocyte developmentin BDNF/ mice. (AF) Thymocytes from 12-to 14-day-old BDNF/ in comparison toBDNF+/+ or BDNF+/ mice were analyzed byFACS analysis. (A) Thymus cellularity, (B) thepercentage of CD4/CD8 DN thymocytes and (C)of DN3-stage CD25+CD44 thymocytes, (D) thecell size of the DN3-stage thymocytes as per-centage of the mean of WT mice in each indi-vidual experiment, (E) the percentage of DN3-stage CD27+ thymocytes and (F) SP CD25+CD4+

thymocytes are indicated. Data are pooled fromat least two independent experiments, withn = 78 BDNF/ and n = 611 BDNF+ micewith the exception of (D) with n = 18 micefor both genotypes. Each animal is representedby a single symbol. Statistical analysis wasperformed using the MannWhitney U testwith ***p < 0.001, **p < 0.01, and *p < 0.05.(G) Representative FACSplots of CD4/CD8 stain-ings (upper panel) and CD25/CD44 stainingsof CD4/CD8 DN thymocytes (lower panel) ofBDNF/ (right) and control BDNF+ mice (left).The percentages on the respective cell pop-ulations are indicated in the quadrants. Thequadrants on the right represent a schematicoverview about the thymocyte subpopulations.Plots are representative of at least five indepen-dent experiments performed.

(Supporting Information Fig. 2). Furthermore, the percentage ofCD4+CD25+ SP cells representing selected SP thymocytes as wellas naturally occurring regulatory T cells (Treg) was not differentbetween BDNF/ and control mice (Fig. 2F). Expression of theactivation marker CD69 was similar in double-positive (DP) andSP thymocytes in mice from all genotypes (Supporting Informa-tion Fig. 3), whereas the ratio of SP CD4 to DP thymocytes was

reduced in BDNF/ mice (data not shown). This finding togetherwith the reduced percentage of SP thymocytes as well as T-cellnumbers in the periphery points to a disturbed maturation fromthe DP to the SP stage. Taken together, complete absence of BDNFin the thymus results in disturbed T-cell development character-ized by massively reduced thymic cellularity and a partial DN3block.

Table 1. Absolute numbers of thymocyte subpopulations in BDNF/ mice in comparison to controlsa)

CD4+ CD8+ DP DN DN1 DN2 DN3 DN4

BDNF+ 12.98 6.2 8.05 6.9 96.9 46.4 8.63 6.4 0.88 0.6 0.61 0.56 3.1 2.3 3.93 3.17BDNF/ 3.12 1.6 1.83 1.6 31.3 19.9 2.71 2.4 0.32 0.26 0.25 0.24 1.28 1.14 0.82 0.81Statistical evaluation *** ** *** *** ** * ** ***Ratio BDNF+/ BDNF/ 4.1 4.4 3.1 3.1 2.75 2.4 2.4 4.8

a)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 (CD44CD25+), and DN4 (CD44CD25) from BDNF-sufficient (BDNF+, n = 16) and BDNF-deficient12- to 14-day-old mice (BDNF/, n = 11). *p < 0.05, **p < 0.01, ***p < 0.001.



Figure 3. Defective thymocyte development in lckCre BDNFfl/fl mice. (AF) Thymocytes from 6- to 8-week-old lckCre BDNFfl/fl mice were analyzedin 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= 48mice 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 percentageson the respective cell populations are indicated in the quadrants. Plots are representative of four independent experiments performed.

T-cell-specific BDNF deficiency recapitulates thephenotype of BDNF/ mice

Since BDNF is expressed in hematopoietic cells of different lin-eages [17], we investigated which cell type is responsible for theproduction of functionally relevant BDNF in the thymus. To thisend, we used conditional KO mice in which BDNF is deleted inimmune cells of different lineages [33]. No differences in thy-mocyte subpopulations could be observed in mice lacking BDNFspecifically in cells of the myeloid lineage (LysMCre BDNFfl/fl,data not shown), pointing to the fact that myeloid cells do notseem to play a major role for BDNF production in the thymus.lckCre BDNFfl/fl mice lack BDNF selectively in thymocytes startingfrom the DN2 stage and in mature T cells. Thymocyte cellular-ity in 6- to 8-week-old adult lckCre BDNFfl/fl mice was signifi-cantly reduced in comparison to control mice carrying two floxedBDNF 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 micewas even more pronounced compared with the situation in con-ventional BDNF/ mice. The percentage of DN thymocytes wasincreased twofold (Fig. 3D and G), corroborating the results fromconventional BDNF/ mice. The partial developmental block pre-viously seen at the DN3 stage in BDNF/ mice (Fig. 3E andG) 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 alsoreflected the data obtained with BDNF/ mice, revealing evenincreased absolute numbers of DN and DN3 thymocytes in lckCreBDNFfl/fl mice compared with controls irrespective of the overallreduced thymic cellularity, which is mirrored in all other subpopu-lations (Table 2). The thymus phenotype could also be observed in2-week-old lckCre BDNFfl/fl mice suggesting that the effect is notage dependent (data not shown).

Thus, selective absence of BDNF in the T-cell lineage causes apartial 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)

CD4+ CD8+ DP DN DN1 DN2 DN3 DN4

BDNF+ 16.63 6.3 5.83 3.2 126.4 43.8 4.85 1.4 0.6 0.2 0.19 0.06 2.49 0.11 2.27 0.63BDNF 8.24 3.9 3.02 1.2 84.6 30.9 5.63 2.5 0.31 0.21 0.15 0.05 3.0 1.8 1.7 1.37Statistical evaluation * * ns ns ns ns ns nsRatio BDNF+/ BDNF/ 2.01 1.93 1.49 0.8 1.93 1.27 0.83 1.33

a)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 (CD44CD25+), and DN4 (CD44CD25) from 6- to 8-week-old lckCre BDNFfl/fl (BDNF) and Crenegative BDNFfl/fl control mice (BDNF+, n = 8 for CD4+, CD8+; DP and DN and n = 4 for DN14). *p < 0.05; ns, not significant.



DP to mature SP T cells and therefore modulates thymocyte devel-opment, mirroring the effects observed in conventional BDNF/

mice. The fact that LysMCre BDNFfl/fl mice do not show theseeffects argues for the T cells themselves being the major source offunctionally relevant BDNF in the thymus.

ERK signaling in DN3 thymocytes is reducedin the absence of BDNF

Several mechanisms could potentially contribute to the partialDN3 block observed for BDNF-deficient thymocytes. First, BDNFhas been described as a neuronal survival factor [34]. In viewof the massive thymocyte apoptosis of functionally deficient TrkBmice [31], and the recently discovered role of BDNF as a survivalfactor for B-cell lines [35], we analyzed the spontaneous apoptosisof BDNF/ and control thymocytes ex vivo. There was no differ-ence in the percentages of AnnexinV+ CD4 SP and DP thymocytescultured for up to 22 h (Fig. 4A and data not shown). DN sub-populations showed a slightly more heterogeneous pattern withBDNF-deficient DN1 to DN3 thymocytes having a lower apoptosisrate (Fig. 4CE), whereas BDNF-deficient DN4-stage thymocytesexhibiting slightly increased apoptosis (Fig. 4F). However, over-all the apoptosis rate of DN thymocytes did not significantly differbetween BDNF-deficient and control cells (Fig. 4B). Similar resultswere obtained for early apoptotic cells (AnnexinV+ 7AAD, datanot shown). These data argue against the possibility that increasedinduction of apoptosis underlies the massive cell loss observed inBDNF/ mice.

Another reason for diminished thymocyte transition from theDN3 to DN4 stage could be disturbances in molecules criticallyinvolved in this process. We therefore analyzed the intracellularexpression levels of the TCR- chain at the DN3 stage and foundno significant differences between BDNF-deficient (from lckCreBDNFfl/fl mice) and control cells (Fig. 5A). Moreover, the expres-sion of the pre-TCR as assessed by immunohistological stainingwas similar (Fig. 5B).

Finally, signaling via the pre-TCR at the DN3 stage could bedisturbed. The analysis of phosphorylated signaling proteins byintracellular FACS staining revealed that there was a significantreduction in pERK expression levels in BDNF-deficient DN3 thymo-cytes compared with controls (Fig. 5C and D) whereas the expres-sion levels of pAKT were identical (Fig. 5F and G). These resultswere corroborated by Western blot analysis of FACS-sorted DN3thymocytes from 13-day-old BDNF/ and control mice. Again,DN3 cells from BDNF/ mice showed a marked decrease inpERK levels whereas pAKT levels were similar (Fig. 5E and H).Of note, there was no difference in pERK expression levels in DPand SP thymocytes between BDNF/ and control mice (Support-ing Information Fig. 4). pAKT levels were very low in DP and SPthymocytes again without any difference between the genotypes(data not shown). The same reduction of pERK expression levels inDN3 thymocytes was observed in 6- to 8-week-old lckCre BDNFfl/fl

mice compared with BDNFfl/fl controls (Supporting InformationFig. 5).

BDNF deficiency does not affect T-cell diversity,functionality, and homeostatic proliferation

After having established that thymocyte selection is affected byBDNF deficiency, we next aimed at investigating its effect onclonal diversity of SP thymocytes and on the survival of periph-eral T cells and/or their homeostatic proliferation. To this end,we FACS-sorted CD4+ SP thymocytes from BDNF/ and con-trol mice, amplified cDNA for six different TCR- families andsequenced the resulting amplicons. There was no difference in theyield of different clones or in the frequency of individual clonesand a striking similarity in both usage and combination of dif-ferent VDJ elements, suggesting a similar level of clonal diversityin both genotypes (Supporting Information Fig. 6). To assess theT-cell capacity of homeostatic proliferation, we purified T cellsfrom either lckCre BDNFfl/fl mice or BDNFfl/fl mice, labeled themwith CFSE, and transferred them into RAG1-deficient mice. Fivedays later, analysis of spleen and lymph nodes revealed similarnumbers of CD4+ and CD8+ T cells of both genotypes, whichhad undergone comparable homeostatic proliferation (SupportingInformation Fig. 7). Similar results were obtained when purifyingand transferring T cells from 2-week-old BDNF/ and appropriatecontrol mice (data not shown).

Finally, we investigated whether the partial block in thymo-cyte maturation as a result of BDNF deficiency would affect theperipheral T-cell function in lckCre BDNFfl/fl mice. First, we stud-ied proliferation and cytokine secretion of CD4+ and CD8+ T cellsafter unspecific stimulation by monoclonal anti-CD3/CD28 anti-bodies. CD8+ T cells from the spleen proliferated more vigorouslythan CD4+ T cells, but there was no difference between the geno-types (Fig. 6A), a finding that could be confirmed using purifiedT cells from the lymph nodes (Fig. 6B). The T cells produced IFN-and IL-17 upon stimulation, the levels again not being dependenton the expression of BDNF (Fig. 6C and D). Second, to ascertainthe result of antigen-specific immune responses, we immunizedlckCre BDNFfl/fl mice and appropriate controls with MOG35-55(MOG ismyelin oligodendrocyte glycoprotein) and analyzed T-cellresponses at day 10 after immunization [33]. Despite the observedT-cell lymphopenia in conditional KO mice, neither T-cell prolif-eration nor production of Th1/Th17 cytokines upon restimulationwith MOG peptide in vitro differed between lckCre BDNFfl/fl andcontrol mice (Fig. 6E and F).

Discussion

Besides their essential functions for development, homeostasis,and regeneration in the CNS, neurotrophins and neurotrophicfactors also have been previously reported to affect cells ofthe immune system. Here, we demonstrate a functional role ofendogenous BDNF for T cells at the level of thymocyte develop-ment. Thymocytes from mice deficient for BDNF in all cells orspecifically in the T-cell lineage displayed a partial block in thy-mocyte development at the DN3DN4 transition and DPSP matu-ration, which presumably resulted in an overall reduction of totaland SP thymocyte numbers without affecting their clonal diversity.



Figure 4. Analysis of spontaneous apoptosisof thymocyte subpopulations. (AF) Thymocytesfrom 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 thepresence of AnnexinV positive cells either directlyex vivo or after 10 and 22 h of culture. The cellswere gated for (A) CD4/CD8DP, (B) CD4/CD8DN thy-mocytes, (C) CD44+CD25 DN1, (D) CD44+CD25+

DN2, (E) CD44CD25+ DN3, and (F) CD44CD25

DN4 thymocytes. The percentage of AnnexinV pos-itive cells is shown as mean SEM, and data arepooled from three independent experiments per-formed. Statistical analysis was performed usingthe MannWhitney U test, only significant differ-ences are shown with **p < 0.01.

One can assume that impaired thymocyte development is the causeof the observed mild peripheral T lymphopenia. However, acuteadaptive immune responses were not compromised as exempli-fied by the unaltered T-cell proliferation and effector cytokine pro-duction both after antigen-independent anti-CD3/CD28-mediatedstimulation or MOG35-55 immunization and antigen-specific recallresponses. Furthermore, it was already previously shown that thecourse of MOG-EAE was not different in lckCre BDNFfl/fl micecompared with controls [33]. We cannot rule out, however, thatbesides these obviously not compromised acute immune responsestested here the defect in thymocyte development and the resultingperipheral lymphopenia may influence chronic immune responses,impair the host response against certain infections or impacton immunological senescence. Further studies have to be per-formed to examine these possibilities over a longer observationperiod.

BDNF has been implicated in axonalmaintenance and neuronalsurvival [10, 34]. It also promotes the survival of nonneuronalcell types such as B-cell lines [35] so that massive lymphocyteapoptosis occurs in the thymus of functionally deficient TrkB mice[31]. Furthermore, survival signals are mandatory for thymocytedevelopment, as revealed in mice deficient for Akt1/Akt2, whichalso display a DN3 block due to increased thymocyte apoptosis

[36]. Therefore, a similar role for BDNF in survival of developingthymocytes is conceivable, and we first investigated the influ-ence of BDNF deficiency on the apoptosis induction of differentthymocyte subpopulations. Surprisingly, we could not detect sig-nificantly increased overall apoptosis in BDNF/ thymocytes, sug-gesting that the induction of apoptosis is not the major cause forthe observed reduction of thymic cellularity. There were some dif-ferences in the apoptosis induction in the DN compartment withDN1 to DN3 thymocytes succumbing to somewhat lower and DN4to somewhat higher rates of apoptosis in BDNF-deficient thymo-cytes, but the differences were small and are probably only of, ifany, minor relevance for the observed partial DN3 block.

The main influence of BDNF deficiency on thymocyte devel-opment was seen for the transition from the DN3 to DN4 stage,a critical step requiring the coordinate action of many differentsignaling molecules. VDJ recombination at the DN3 stage leadsto the expression of the TCR- chain, which pairs with the pre-TCR chain conveying survival, proliferation, and transition to theDN4 stage. These processes critically depend on the recombina-tion machinery eventually leading to the expression of the TCR-chain as well as components of the signaling cascade deliveredby the pre-TCR such as lck/fyn, Zap70/syk, and LAT [37]. Wetherefore tested whether the lack of BDNF impaired the



Figure 5. pERK but not pAKT levels are reducedin BDNF/ DN3 thymocytes. (A) Thymocytesfrom lckCre BDNFfl/fl and BDNFfl/fl control mice(n = 5) were surface-stained for CD4, CD8, CD25,and CD44 and intracellularly for TCR- chainexpression. The cells were first plotted for CD4against CD8, the DN population was plotted forCD44 against CD25, and the CD44CD25+ (DN3)population was considered for further analysis.Histograms referring to TCR- chain expression inthe DN3 subpopulation are shown, and the per-centage of positive cells is indicated. Data are rep-resentative of three independent experiments. (B)DN3 thymocytes from 12-day-old BDNF+/+ control(left) and BDNF/ (right) mice were sorted, platedon coverslips by cytospin and stained for pre-TCRby immunohistochemistry. Bar = 20 m. Arrowspoint to positive cells. (CH) Thymocytes from 12-to 14-day-old BDNF/ and BDNF+/+ control micewere stained for CD4, CD8, CD25, and CD44 andintracellularly for pERK or pAKT. Representativehistograms of pERK (C) or pAKT (F) stainings of DN3thymocytes of control (dotted line) and BDNF/

mice (solid line) are shown. The filled histogramrepresents the control staining without primaryantibody. Quantification of pERK (D) or pAKT (G)stainings (geometric mean SEM) is indicated.Data shown are from n = 48 mice/group andare pooled from at least two independent exper-iments. Statistical analysis was performed usingthe MannWhitney U test with *p < 0.05. (E and H)Protein lysates from sorted DN3 thymocytes wereanalyzed for pERK (E) or pAKT (H) by Western blot.Protein loadingwas controlledwith ERK1/2 or actinantibodies. Numbers beneath the blots indicate therelative expression level of pERK and pAKT afterquantification and relation to protein loading con-trols. Blots are representative of two independentexperiments performed.

expression or function of some of those molecules. We did notfind differences in the expression levels of the TCR- chain orthe pre-TCR, but BDNF directly impacts a critical signaling path-way implicated in pre-TCR signaling. There are many signalingpathways downstream of the high-affinity BDNF receptor TrkBthat are shared with pre-TCR signaling such as ERK and AKTsignaling [38]. We tested the phosphorylation status of AKTand ERK and found that pERK levels were indeed reduced inBDNF-deficient DN3 thymocytes whereas pAKT levels were notaffected. This suggests that BDNF could directly modulate thy-mocyte differentiation via modulating pERK levels in differentiat-ing thymocytes. Interestingly, mice deficient in ERK1 also show adefect in thymocyte maturation resulting in decreased SP CD4+

and CD8+ populations [39]. Diminished pERK levels were onlyobserved in the DN3 subpopulation but not in DP and even more

mature SP thymocytes, suggesting that BDNF deficiency influ-ences thymocyte maturation only in a very limited window oftime and/or space. Additionally, BDNF may exert its effects onthymocytes via activation of cAMP response element-binding pro-tein (CREB), a transcription factor phosphorylated and activatedby pre-TCR signaling via PKC- and ERK-MAPK-mediated path-ways [40]. Interestingly, CREB/ mice show a similar pheno-type 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-2production are diminished inmice expressing a dominant-negativeform of CREB [42]. CREB is a major mediator in the BDNF signal-ing cascade [43]. Therefore, it is tempting to speculate that BDNFcould feed into the CREB-mediated signaling cascade required forthe development of DN thymocytes.



Figure 6. Peripheral T lymphopenia in lckCreBDNFfl/fl mice does not influence T-cell func-tion. (AD) T cells from lckCre BDNFfl/fl miceand BDNFfl/fl littermates were purified, labeledwith CFSE, and cultured with monoclonal anti-CD3/CD28 antibodies for 3 days. The percent-age of proliferating CD4+ or CD8+ T cells fromthe (A) spleen or (B) lymph node of lckCreBDNFfl/fl mice and BDNFfl/fl littermate controlsis depicted. There was no proliferation in theabsence of anti-CD3/CD28 antibodies (data notshown). In parallel experiments, the secre-tion of (C) IFN or (D) IL-17 from either anti-CD3/CD28 stimulated or unstimulated purifiedT cells from the spleen after 3 days of cul-ture was determined. Data are presented asmean SEM (n = 5) and are pooled fromtwo independent experiments. Statistical anal-ysis was performed using the MannWhitneyU 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. Tendays later, splenocyte primary cultures wereanalyzed for (E) T-cell proliferation and (F)IFN- as well as IL-17 production in response toMOG35-55 or polyclonal stimulation with ConA.Data are presented as mean SEM (n = 34mice/group) and are representative of two inde-pendent experiments. Statistical analysis wasperformed using the MannWhitney U test anddid not reveal significant differences betweenlckCre BDNFfl/fl and control mice.

Lack of BDNF impacted on thymocyte development and pERKexpression levels in the DN3 subpopulation irrespective of whetherit is confined to T cells or ubiquitously absent. This observationfavors the notion of T-cell-derived BDNF [26] acting in a paracrineor autocrine fashion being responsible for the observed pheno-type. Since BDNF is already expressed in DN thymocytes (datanot shown), these two possibilities cannot be distinguished by thecurrent approach. In contrast, BDNF from other potential sources,such as thymic macrophages, apparently plays no or only a minorrole as evident by the lack of a thymic phenotype in LysMCreBDNFfl/fl mice, which lack BDNF in myeloid cells. That the BDNFpotentially produced by epithelial cells plays a role in thymocytedevelopment could not consequently be ruled out but seems ratherunlikely. 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 thematuration of conventional thymocytes was partially impairedat the DN3 stage, suggesting a differential dependence on BDNFfor different T-cell subsets.

One important question is the extent and the consequencesof the distortion of T-cell development by the absence of BDNF.The partial DN3 block was evident in every setting and at everyage of the mice and, even if it was far from complete, one canassume that it certainly impacts the thymic output with conse-quences for T-cell cellularity in the periphery, a compartment thatnormally reacts in a very plastic manner to subtle changes in thy-mocyte development and output. The effect on maturation of DPto SP thymocytes was more evident in adult lckCre BDNFfl/fl mice,which suggests that this maturation block is able to enhance theeffect of the partial DN3 block on thymic output leading eventually



to peripheral T lymphopenia. Whether a partially defective posi-tive selection or enhanced negative selection is responsible for theeffect on the maturation of DP to SP thymocytes and the relativecontribution of the partial DN3 and DP maturation block to theoverall phenotype remains to be elucidated in further studies. Insummary, regarding BDNF as a factor contributing to efficient thy-mocyte development extends our understanding of the pleiotropicnature of this neurotrophin beyond its well-established role in thenervous system.

Materials and Methods

Animal experimentation

C57Bl/6 mice were purchased from Charles River (Sulzfeld,Germany). BDNF/ mice were generated at the Max-Planck Insti-tute for Neurobiology, Martinsried, Germany, using D3, 129Svembryonic stem cells [44] and subsequently backcrossed to theC57Bl/6 background for more than 15 generations. ConditionalBDNFfl/fl mice were backcrossed to the C57Bl/6 background formore than ten generations [45] and were crossed with miceexpressing Cre under the control of the proximal lck promoter[46] or the LysM promoter [47]. All control mice were BDNF+ orBDNFfl/fl littermates. All experiments were approved by the LowerSaxony state authorities for animal experimentation.

FACS analysis

FACS analysis was performed using a FACSCalibur or FACS AriaII sorp from BD Biosciences (Heidelberg, Germany) and CellQuestsoftware as previously described [48]. The following antimouseclones from BD were used: CD4 (H129.19 or GK 1.5), CD8a (53-6.7), CD3 (145 2C11), CD25 (7C4), CD44 (IM7), CD62L (Mel14),CD69 (H1 2F3), NK1.1 (PK136), and TCR (B1). For thymusanalysis, cells were routinely plotted for CD4 against CD8, and theCD4CD8 population for CD44 against CD25. The CD44CD25+

(DN3) population was subjected to further analysis. For periph-eral lymphoid organs, cells were plotted for CD4 against CD8, andthe CD4+ and CD8+ populations were analyzed separately (thegating strategy is shown in Supporting Information Fig. 8). Forintracellular staining, the FoxP3 staining kit (clone FJK-16s) fromeBioscience (San Diego, CA, USA) was used according to the man-ufacturers instructions. Intracellular staining for the TCR- chainwas performed using Fix-Perm (BD Biosciences) and anti-TCR(H57-597, BD Biosciences). For the intracellular staining of phos-phorylated signaling proteins, thymocytes were incubated withanti-CD3 (clone 145-2C11, eBioscience), crossed-linked for 10minat 37C, fixed with Fix-Perm (BD Biosciences), and subsequentlystained for CD4, CD8, CD25, CD44 and Alexa488-conjugatedpAKT (Cell Signalling Technology, Danvers, MA, USA) or pERKTyr 204 (Santa Cruz Biotechnology, Dallas, TX, USA) followedby goat-anti-rabbit APC conjugated secondary antibody (Dianova,Hamburg, Germany).

For FACS analysis of peripheral immune cells, cells were gatedon living lymphocytes, followed by gating for CD4+ cells, andsubsequently analysis for the appropriate markers was performed.For thymus analysis, cells were gated on living lymphocytes, fol-lowed by discrimination for CD4 and CD8. Cells negative for bothmarkers were analyzed for CD25 and CD44 to allow definition ofthe four DN subsets. In some cases, CD25+CD44 DN3 cells weregated and analyzed for the appropriate markers.

CFSE labeling and cell transfer

T cells isolated from the spleen of 13-day-old BDNF/ orBDNF+/+ littermates were purified using a pan-T-cell isolation kit(Miltenyi, Bergisch-Gladbach, Germany) and labeled with CFSEas previously described [49]. A total of 1 107 cells were adop-tively transferred intravenously into RAG1/ mice. Five dayslater, spleen and lymph node cells from the recipient mice wereanalyzed by FACS staining for CD3, CD4, and CD8 expression.

Thymocyte apoptosis

Thymocytes were collected from either 13-day-old BDNF/ miceor control littermates and cultured for up to 22 h in RPMI/10%FCS. The apoptosis rate was determined by flow cytometry usingFITC-labeled AnnexinV (BioLegend, San Diego, CA, USA) in com-bination 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) from12-day-old BDNF-deficient and control mice and plated on coverslips using a Shandon Cytospin 4 (Thermo Scientific, Dreieich,Germany). Cells were stained via immunocytochemistry employ-ing the M.O.M. Kit (Vector via Linaris, Dossenheim, Germany)with a mouse anti-pre-TCR alpha Ab (1:100; BD Bioscience) andwith anti-mouse Alexa 488 as secondary antibody (1:1000) andantifade reagent with DAPI (all Invitrogen, Darmstadt, Germany).

Western blot

Protein extracts from FACS-sorted DN3-stage thymocytes wereprepared as described [50]. Proteins were run on a 10% SDS-PAGE, transferred to nitrocellulose, and analyzed for the expres-sion of pERK1/2 (Thr202/Tyr204), pAKT (Ser473), total ERK1/2 (allfrom Cell signalling Technology), and -actin (Santa Cruz Biotech-nology). Primary antibodies were detected with mouse-anti-rabbitantibodies (Jackson ImmunoResearch Laboratories, West Grove,PA, USA) and the ECL detection system (Thermo Scientific Pierce,Rockford, IL, USA). Quantification of protein expression was per-formed with Kodak software.



In vitro T-cell culture and ELISA

T cells from young adult lckCre BDNFfl/fl mice or BDNFfl/fl miceas controls were purified using a negative selection purificationkit (Easy sep mouse T-cell isolation kit, Stemcell technologies,Grenoble, France), labeled with CFSE and plated in 24-well platesat a concentration of 2106 cells/mL and incubated with 1 g/mLanti-CD3 (145-2C11) and 5 g/mL anti-CD28 (37.51; both fromeBioscience). After 3 days, cells were stained for CD4 and CD8 andanalyzed by FACS analysis. At the same time, the supernatant wascollected and analyzed for IFN-, and IL-17 levels by sandwichELISA, as previously described [51]. mAb pairs and recombinantcytokine standards were purchased from BD for all cytokines.

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+ SPthymocytes FACS-sorted using the FACS Aria II sorp. RNA wasprepared using the RNeasy Micro Kit (Qiagen, Venlo, The Nether-lands) and reverse transcription was performed using the First-strand cDNA Synthesis Kit (Thermo, Waltham, MA, USA). For theamplification of TCR- alleles, primers specific for six differentTCR- families (V2, V3.1, V7, V8.1, V8.2, and V11) wereapplied in combination with a reverse primer from the C region asdescribed previously [52]. PCR products from each single mousewere pooled and sequenced using an Illumina Miseq instrumentwith 2 150 bp paired-end read configuration. After basecalingand demultiplexing individual samples using the Illumina CASAVApipeline, the reads were assembled with the PEAR software [53].Unassembled and low-quality reads were disregarded from fur-ther downstream analysis. The number of assembled reads variedbetween 787 351 and 2 170 962 (1 320 518 462 782) persample with an average read length of 179 33 bp. To identifyV/D/J rearrangements, the sample files were analyzed using theVidjil software [54]. For this purpose, germline data of knownV/D/J elements for Mus musculus were downloaded from theIMGT database (www.imgt.org, as of December 2014). Analysis ofrecombination yielded on average 18 333 8606 clones per sam-ple (13 820 4601 per million reads, see Supporting InformationFig. 6). For combined data from both genotypes, the total numberof detectable clones was 53505/56022 (control/BDNF/).

Induction of active MOG-EAE

For active induction of EAE, mice received an s.c. injection atflanks and tail base of 50 g MOG3555 peptide (Charite, Berlin,Germany) as previously described [55].

3[H] thymidine proliferation assay

Splenocytes were prepared 9 days after immunization of miceand cultured with media alone or in the presence or absence of

20 g/mL of MOG 3555 peptide or 1.25 g/mL ConA. Prolif-eration was investigated by measuring 3[H]-thymidine uptake asdescribed [52]. Supernatants from a parallel culture were har-vested after 3 days and analyzed for IFN- and IL-17 levels bysandwich ELISA, as described [51].

Statistical analyses

Statistics were performed after controlling for normal distribu-tion by MannWhitney U test (PRISM software, Graph Pad, SanDiego, CA, USA). Data are given as mean SEM. The followingp-values were considered as significant: *p < 0.05, **p < 0.01,***p < 0.001. p 0.05 was considered as not significant.

Acknowledgments: The authors thank M. Weig, B. Curdt, A.Bohl, N. Meyer, and S. Seubert for excellent technical assis-tance; N. Kruse for help with the real-time PCR; and Cathy Lud-wig for language corrections. This work was supported by theGemeinnutzige 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 Sax-ony (to A.F. and F.L.) and by grants from the Deutsche Forschungs-gemeinschaft (FOR 1336 to A.F., SFB-TRR 43 to A.F. and F.L., SFB854, TP 9 to U.B.) and the Bundesministerium fur Bildung undForschung (UNDERSTAND MS to A.F.).

Conflict of interest: The authors declare no financial or commer-cial conflict of interest.

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Abbreviations: BDNF: brain-derived neurotrophic factor DN: doublenegative SP: single positive DP: double positive MOG: myelin oligo-dendrocytes glycoprotein

Full correspondence: Dr. Fred Luhder, Department ofNeuroimmunology, Institute for Multiple Sclerosis Research, TheHertie Foundation and MPI for Experimental Medicine, University ofGottingen, Waldweg 33, 37073 Gottingen, Germanye-mail: [email protected]

Current address: Jens van den Brandt, University of Greifswald MedicalSchool, Central Core & Research Facility of Laboratory Animals,Walther-Rathenau-Str. 49a, 17489, Greifswald, Germany

Received: 2/7/2014Revised: 18/12/2014Accepted: 22/1/2015Accepted article online: 27/1/2015


linker et al-2015-european journal of immunology

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