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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2012 Sep 12;33(1):37–47. doi: 10.1038/jcbfm.2012.128

Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO

Tobias Stubbe 1, Friederike Ebner 1, Daniel Richter 1, Odilo Randolf Engel 2, Juliane Klehmet 2, Georg Royl 2, Andreas Meisel 2, Robert Nitsch 3, Christian Meisel 4, Christine Brandt 4,*
PMCID: PMC3597367  PMID: 22968321

Abstract

Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3EGFP transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3+ Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4+ cells depleted of Foxp3+ Tregs into RAG1−/− mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25+ Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.

Keywords: focal ischemia, immunology, leukocytes, macrophages, microglia, neuroprotection, T cells

Introduction

The innate immune response mediates a cascade of immune events in the lesioned brain after stroke. The brain resident microglia are activated, turn into phagocytes and begin secreting pro-inflammatory cytokines.1 Within hours after ischemia, peripheral leukocytes trespass brain vessels and accumulate in the brain, leading to substantial secondary damage.1, 2, 3 Early detrimental effects of T cells are not related to adaptive immune mechanisms, such as antigen recognition or costimulatory pathways.3 At the same time, susceptibility to bacterial infection is increased by a systemic immunodepression.4 While the latter may be a means of protecting the brain from autoimmunity resulting from the compromised blood–brain barrier,5, 6 the increased risk of infection and systemic inflammation after stroke modulates the immune response to CNS antigens and can worsen functional outcome after middle cerebral artery occlusion (MCAO).7 Whether an adaptive immune response develops after stroke remains unclear and to date, we only know that after disruption of the blood–brain barrier, antigens from the central nervous system, which are physiologically compartmentalized from the periphery, are recognized by the immune system.8 Antigen presentation can lead to different stages of immune response: (1) full activation (Th1 response), (2) ignorance (no costimulation), (3) Th2/3 immune response, or (4) activation or induction of regulatory T cells (Tregs). The latter are highly effective in inhibiting proliferation and cytokine release of lymphocytes and thus contribute to regulatory immune mechanisms.9

Regulatory T cells were recently shown to contribute to neuroprotection after MCAO by secreting interleukin-10 in the brain and antagonizing tumor necrosis factor-α and interferon-γ production.10 Additionally, the group of Kyra J Becker demonstrated that mucosal administration of myelin basic protein can lead to the induction of a transforming growth factor-β-mediated Treg response, resulting in a better outcome for up to 1 month after MCAO.11 However, in a follow-up study, the same group showed that this effect is reversed at 3 months after MCAO, with a Th1 response developing to myelin basic protein.12 As despite these recent insights, little is known about the distribution, induction, and activation of Tregs after MCAO, our study sought to analyze the distribution of Tregs in the brain and in peripheral lymphatic organs up to 30 days after an ischemic insult. In relation to this analysis, we took a close look at the function of CD25+ Tregs in the late phase after MCAO. We also examined the distribution of potential antigen presenters such as microglia, dendritic cells (DCs), and macrophages in the postischemic brain and investigated whether the presence of Tregs is the result of de novo induction or proliferation.

Materials and methods

Animals

Foxp3EGFP reporter mice (C57BL/6J background) were provided by B Malissen.13 RAG1−/− and C57BL/6J were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were bred and maintained under specific pathogen-free conditions at the animal facility of Charité—Universitätsmedizin Berlin. All animal experiments were performed according to the national regulations (German Animal Welfare Act) and institutional guidelines. All animal experiments were approved by the ‘Landesamt fuer Gesundheit und Soziales' in Berlin, Germany.

Middle Cerebral Artery Occlusion

Focal cerebral ischemia was induced by left MCAO using a modified protocol as described previously.14 Mice were anesthetized with isoflurane (Abbott, Abbott Park, IL, USA) 1.5% to 2% v/v oxygen. The left carotis communis artery was exposed through a midline neck incision. A 6-0 silicon-coated nylon monofilament (Serag Wiessner, Naila, Germany) with a heat thickened cone was inserted over the left carotis communis artery into the internal carotid artery and led into the MCA. The monofilament was left for 30 minutes in the MCA until reperfusion. In sham-operated animals, a silicon-coated nylon monofilament was withdrawn immediately after reaching the MCA to avoid ischemia. Lesion volumes were measured 4 to 6 days after MCAO by T2-weighted magnetic resonance imaging (MRI) on a 7-T Bruker scanner (Pharmascan 70/16 AS, Bruker Biospin, Ettlingen, Germany). The delineable hyperintense lesion volume was determined on 20 consecutive coronal slices with 500 μm thickness using Analyze 5.0 (AnalyzeDirect, Overland Park, KS, USA) and the lesion volume was edema-corrected.15 Mice without an edema formation in the cerebral cortex or a lesion volume <25 mm3 were excluded from further evaluation.

Preparation for Fluorescence Activated Cell Sorting Analysis

Middle cerebral artery occlusion was performed and 7, 14, and 30 days later cell suspensions from lymph nodes (LN), spleen, blood, and brain tissue from C57BL/6J mice were analyzed using BD LSR II and FACSCanto II (BD Biosciences, San José, CA, USA). Data were evaluated using FlowJo version 8 (TreeStar, Ashland, OR, USA). Animals were perfused under deep anesthesia with 100 mL of 0.9% saline. After removing the cerebellum, the brain was divided into ischemic ipsilesional and nonischemic contralesional hemispheres. The brain hemispheres were chopped, incubated for 30 minutes at 37°C with 1 mg/mL Collagenase VIII (Sigma Aldrich, St Louis, MO, USA) and then squeezed through a 70-μm cell strainer (BD Biosciences). The brain suspensions were suspended in 35% Easycoll (Biochrome, Berlin, Germany) solution and afterwards transferred on 70% Easycoll in 15 mL tubes. After centrifugation for 30 minutes at 300 g, cells were obtained from the intermediate phase and stained for FACS analysis. The Collagenase VIII step was skipped in experiments using anti-Foxp3 and anti-Ki-67. Cervical LN, inguinal LN, and spleen were isolated, squeezed through a 70-μm cell strainer and erythrocytes were removed by using lysis buffer (1:9 of 0.17 M Trisbase/0.16 M NH4Cl) for 5 minutes at room temperature and subsequently washed. Blood was taken from the left ventricle during the perfusion procedure and transferred into phosphate-buffered saline containing 2 mmol/L EDTA (Carl Roth, Karlsruhe Germany). PBMCs were isolated using Histopaque-1083 gradient (Sigma Aldrich).

Fluorescence Activated Cell Sorting Antibodies

The following monoclonal antibodies were used for flow cytometry staining: CD11b (M1/70), CD11c (HL3), CD4 (RM4-5), major histocompatibility complex class II (MHCII) (M5/114.15.2), T cell receptor (TCR)-β chain (H57-597), CD45 (30-F11), CD8a (53-6.7), CD45RB (16A), all BD Biosciences, CD25 (PC61, Invitrogen, Carlsbad, CA, USA), and CD25 (7D4, Miltenyi Biotech, Bergisch, Gladbach, Germany). Foxp3 (FJK-16s, eBioscience, San Diego, CA, USA) and Ki-67 (B56, BD Biosciences), were used according to the manufacturer's protocol.

Immunohistochemistry

Foxp3EGFP reporter mice were used for immunohistochemistry at days 7, 14, and 30 after MCAO. For this, anesthetized animals were perfused as described above. The brain and spleen were removed and postfixed overnight in 4% paraformaldehyde. For cryoprotection, organs were incubated overnight with 10%, 20%, and 30% sucrose solution. Organs were snap-frozen in 2-methylbutane on dry ice and 12 μm cryostat frontal sections of the brain were prepared. As primary antibodies CD4 (RM4-5) (BD Biosciences), MHCII (M5/114.15.2), biotin CD11c (N418) (eBioscience), rabbit polyclonal to GFP (green fluorescent protein) and chicken polyclonal to GFP (Abcam, Cambridge, UK), rabbit polyclonal to IBA1 (Wako Chemicals, Osaka, Japan) and chicken polyclonal to laminin (Sigma Aldrich) were used. Secondary antibodies and streptavidin, both conjugated with Alexa-Fluor dyes, were purchased from Invitrogen. All slices were stained with DAPI (Roche, Basel, Switzerland). Slices were imaged on a Leica TCS-SL (Wetzlar, Germany) and a Zeiss LSM5 Exciter confocal microscope (Jena, Germany). Spleen sections were used as positive controls and contralesional hemispheres were used as negative controls. Crossreactivity with target tissue could be excluded by staining cryosections with secondary antibodies only. Rat IgG2b K (eBioscience) was used as isotype control for MHCII and Armenian Hamster IgG Biotin (eBiosicence) was used as isotype control for CD11c. Both controls showed no positive reactivity in ischemic hemispheres.

TaqMan Assay

RNA was isolated from whole hemispheres of MCAO-treated mice and sham control. Total RNA (1 μg) was reverse transcribed (QuantiTect Reverse Transcription Kit, Qiagen, Valencia, CA, USA). Interleukin-2 (IL-2) and HPRT were analyzed using TaqMan gene expression assay (Applied Biosystems, Carlsbad, CA, USA). All amplification reactions were performed on ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems). Expression of the target gene IL-2 was normalized with the housekeeping gene HPRT and is displayed as 2−ΔCT.

Adoptive Transfer of Purified T and B Cells

CD4+ cells from LN and spleen of Foxp3EGFP mice were isolated by negative selection using a magnetic cell separation kit from Miltenyi Biotec and applying the manufacturer's protocol, resulting in CD4+ cell purities of over 90%. Afterwards, cells were stained for CD45RB and sorted using a FACSAria cell sorter (BD Biosciences) for CD4+/CD45RBhigh cells depleted of Foxp3–EGFP+ cells. CD45RBhigh cell purity in the CD4+ subset was over 99% with a Foxp3–EGFP+ fraction below 0.035%. B cells were separated from LN and spleen of C57BL/6J mice using a negative B cell separation kit from Stemcell Technologies (Vancouver, Canada) and applying the manufacturer's protocol. The resulting purity was over 97%. One day before MCAO or sham surgery, 5 × 106 naive CD4+/CD45RBhigh cells and 1 × 107 B cells were injected intravenously into the tail vein of RAG1−/− mice. In all, 14 days after surgery, mice were killed and the brain, LN, blood, and spleen were analyzed by fluorescence activated cell sorting (FACS) as described above.

Middle Cerebral Artery Occlusion with Depletion of CD25+ Regulatory T Cells

Middle cerebral artery occlusion was performed using adult Foxp3EGFP reporter mice as described above. For in vivo depletion of CD25+ Tregs, 250 μg anti-CD25 (clone PC61) and for the control group 250 μg isotype, both Biolegend (San Diego, CA, USA), were injected intraperitoneally on days 3 and 14 after MCAO. Magnetic resonance imaging was performed on days 3 and 27 after MCAO. On day 3, MRI lesion volumes were determined such as described above and animals with a lesion volume <15 mm3 were excluded. On day 27, lesion volumes were determined indirectly by measuring atrophy and scarred tissue.

Functional outcome was assessed 14 and 27 days after MCAO by gait analysis as described elsewhere.16 We used an automated computer-assisted method (CatWalk, Noldus Information Technology, Wageningen, The Netherlands) according to the manufacture's instructions and published procedures.17 In brief, animals were trained two times to walk over an elevated 1.3 m long glass plate illuminated from the side, with the home cage serving as bait. Contact of animal paws with the glass plate lead to changed refractive index of the internally reflected fluorescent light, lighting up the paw's contact area and being detected by a high-speed camera underneath the glass plate for analysis. We acquired a minimum of three compliant runs, which had to fulfill minimum run duration of 0.5 seconds, maximum run duration of 5 seconds, and a maximum speed variation of 60%. Runs, where animals turned or walked backwards, were not used for analysis. Also, runs in which the software was unable to calculate phase dispersion due to, for instance, anchor paw being undetected, were excluded from statistical analysis.

At day 30 after MCAO, animals were prepared for FACS analysis. Additionally, blood was withdrawn on day 9 from the cheek and analyzed by FACS to determine efficiency of Tregs depletion.

Statistical Evaluation

We used GraphPad Prism version 5.0 (La Jolla, CA, USA) and SPSS version 15.0.1 (SPSS, Chicago, IL, USA) for statistical data analysis. Unpaired Student's t-test was used to compare MCAO groups with the respective sham control and to compare data from anti-CD25-treated mice with isotype control mice. For comparisons of ischemic hemispheres with contralesional hemispheres, paired Student's t-test was applied. One-way ANOVA (analysis of variance) followed by post hoc test Games–Howell was used for pairwise multiple comparisons of ischemic hemispheres from days 7, 14, and 30 after MCAO. One-way ANOVA followed by post hoc test Dunnett's was used to compare lymphatic organs and blood with the ischemic hemisphere.

Results

Regulatory T Cells Showed Prolonged Accumulation in the Ischemic Hemisphere

We first examined the distribution of CD4+ cells and Tregs at days 7, 14, and 30 after MCAO using FACS technology. Cells were gated for CD45high lymphocytes, which were distinguished from the more granular macrophages and DCs by the side scatter. The lymphocytes were then gated to identify the CD11b−/TCR+ population and subdivided into CD4+ and CD8+ subsets. CD4+ Tregs were specified by the expression of Foxp3 (Figure 1A).

Figure 1.

Figure 1

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Flow cytometry analysis of brain hemispheres and lymphatic organs for CD4+ cells and CD4+/Foxp3+ regulatory T cells (Tregs) at days 7, 14, and 30 after middle cerebral artery occlusion (MCAO). (A) Example of successive gating shown on cells from ipsilesional hemisphere 14 days after MCAO. (B) Count of CD4+ cells and (C) count of Foxp3+ Tregs isolated from whole hemispheres. (D) Comparison of the proportion of Foxp3+ Tregs in the CD4+ subset between the postischemic hemispheres, cervical lymph nodes (LN), and inguinal LN. (E) Comparison of the proportion of Foxp3+ Tregs in cervical LN and inguinal LN with sham-operated animals 14 days after surgery. Data are presented as mean±s.d. and n=4 to 12 per group. *P<0.05; **P<0.01; ***P<0.001.

An increase of CD4+ cells was found in ischemic hemispheres on days 14 and 30 compared with the contralesional site and sham-operated animals and on day 7 compared with the contralesional site (Figure 1B). An increase of Foxp3+ Tregs was observed in ischemic hemispheres on days 7, 14, and 30 compared with contralesional and sham control hemispheres (Figure 1C). Additionally, the number of CD4+ cells and Foxp3+ Tregs was significantly higher in ischemic hemispheres on days 14 and 30 compared with day 7 (Figures 1B and 1C). At the same time, the proportion of Foxp3+ Tregs in the CD4+ population was significantly elevated ipsilesionally on days 14 and 30 compared with day 7 and peripheral lymphatic organs (Figure 1D). Regulatory T cells were increased in cervical LN, inguinal LN (Figure 1E), and spleen (data not shown) compared with sham-operated animals only on day 14.

To investigate the distribution of CD4-positive cells and Foxp3-positive Tregs in the ischemic brain, we stained coronal brain cryosections from Foxp3EGFP reporter mice 7, 14, and 30 days after MCAO using antibodies against CD4, GFP, and laminin. CD4-positive cells and CD4/GFP-positive Tregs were found in the peri-infarct area and in the infarct area and had trespassed laminin stained brain vessels 14 days after MCAO (Figures 2A and 2B). On day 7 after reperfusion, only few CD4-positive cells and CD4/GFP-positive Tregs were detected in coronal brain sections in the ischemic hemisphere. However, we then found a strong accumulation of CD4-positive cells and CD4/GFP-positive Tregs in the infarct area 14 days and 30 days after MCAO (Figures 2A and 2C), confirming the FACS data described above.

Figure 2.

Figure 2

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Microscopic localization of CD4/GFP (green fluorescent protein)-positive regulatory T cells (Tregs) in postischemic hemispheres from Foxp3EGFP mice. (A) Light microscope image of 12 μm coronal brain cryosection, showing a section of the ischemic hemisphere at day 14. Arrow indicates morphologically conserved, peri-infarct area, while the asterisk indicates the infarct area with loss of morphological integrity and a highly granular appearance. (B) Confocal image taken 14 days post-middle cerebral artery occlusion (dpMCAO). Laminin-positive vessels are shown in blue. CD4-positive (red) and GFP-positive (green) Tregs trespassed vessels in peri-infarct area. (C) Accumulation of CD4-positive cells and GFP-positive Tregs in the infarct area.

Microglia, Macrophages, and Dendritic Cells Accumulate in Postischemic Hemispheres

To analyze the amount and distribution of microglia, DCs, and macrophages, we performed FACS staining of brain isolated cells at days 7, 14, and 30 after MCAO. CD45 has been used previously as a marker for the identification of CD11b+ microglia, expressing low levels of CD45, and CD11b+ macrophages/DCs, expressing high levels of CD45, after ischemic stroke2, 18, 19 and brain lesion.20 In brain isolates from ischemic hemispheres, a slight shift in the expression of CD45 and SSC-A was detected in CD45dim microglia at days 7, 14, and 30 after MCAO. Nevertheless, microglia were identified as a distinct subset in the CD45 versus SSC-A dot plot throughout the study (Supplementary Figures 1A and B). We gated at the CD45high population containing CD11b+/CD11c− macrophages and CD11b+/CD11c− DCs (Figure 3A) and at the CD45dim/CD11b+ microglia and determined the number of MHCII+ cells (Figure 3A). Microglia were not increased on day 7 in the ipsilesional hemisphere compared with the contralesional hemisphere, but had approximately doubled by days 14 and 30 (Figure 3B). Importantly, activated MHCII+ microglia were evident on days 14 and 30 in the ipsilesional hemisphere compared with contralesional hemisphere (Figure 3C). An increased accumulation of DCs was observed on days 7, 14, and 30 after MCAO, as were, to a significant extent, macrophages in the ischemic hemisphere at days 14 and 30 compared with the contralateral hemisphere (Figure 3B). MHCII+ DCs were increased on days 7 and 14 in the ischemic hemisphere (Figure 3C). On each analyzed day, the number of MHCII+ macrophages was significantly increased compared with the contralesional hemisphere (Figure 3C).

Figure 3.

Figure 3

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Flow cytometry analysis of microglia, macrophages, and dendritic cells (DCs) from the brain 7, 14, and 30 days after middle cerebral artery occlusion (MCAO). (A) Example of successive gating shown on cells from the ipsilesional hemisphere 14 days after MCAO. Microglia were CD45dim/CD11b+ cells. DCs were CD45high/CD11b+/CD11c+ cells and macrophages CD45high/CD11b+/CD11c−. (B) Comparison of microglia, DCs, and macrophages isolated from whole hemispheres at days 7, 14, and 30 after MCAO. (C) Comparison of MHCII+ microglia, DCs, and macrophages at days 7, 14, and 30 after MCAO. Data are presented as mean±s.d. and n=3 to 5 per group. *P<0.05; **P<0.01.

MHCII Expressing Cells Contact with Foxp3-Positive Regulatory T Cells

We next analyzed the distribution of microglia, macrophages, and DCs within the ischemic hemisphere in combination with MHCII expression in Foxp3EGFP reporter mice. Here, we used CD11c as a specific marker for DCs and Iba1 for cells from the monocytic lineage including microglia and macrophages. We detected Iba1/MHCII-positive cells on each day in the infarct area. On days 14 and 30 after MCAO, Iba1-positive and Iba1/MHCII-positive cells were found at higher amounts than on day 7, which was in line with the increased number of microglia seen in the FACS analysis (Figures 3B and 3C and 4A). MHCII expressing cells were not detected in the contralateral hemisphere at any time (data not shown). Dendritic cells, some of which expressed MHCII, were found grouped together, instead of equally distributed, in the infarct area on days 7, 14, and 30 (Figure 4B). Using costaining of GFP with either Iba1/MHCII or CD11c/MHCII, we could clearly show that GFP-positive Tregs contact both Iba1/MHCII-positive cells and MHCII-positive DCs (Figures 4C and 4D).

Figure 4.

Figure 4

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Microscopic localization of Iba1-positive, CD11c-positive, and MHCII-positive cells at days 7, 14, and 30 after middle cerebral artery occlusion (dpMCAO) in infarct area. (A) Iba1-positive and Iba1/MHCII-positive cells. (B) CD11c-positive and CD11c/MHCII-positive dendritic cells (DCs). (C) and (D) GFP (green fluorescent protein)-positive regulatory T cells (Tregs) in contact with MHCII on Iba1-positive and CD11c-positive cells 14 days after MCAO.

Regulatory T Cells Proliferate Strongly in the Ischemic Hemisphere

We next determined whether a proliferation of Tregs occurs after MCAO. At days 7, 14, and 30 after MCAO, we stained lymphocytes isolated from the brain, LN, and spleen using an antibody against Ki-67, as the latter is known to be exclusively expressed in proliferating cells and is present in all phases of the cell cycle.21 Using the same gating as shown in Figure 1A, we found an increased proportion of Foxp3+/Ki-67+ cells in the ipsilesional hemisphere at days 7 and 14, which dropped to almost normal levels by day 30 (Figure 5A). No changes were found in cervical LN, inguinal LN (Figure 5B), spleen, and blood (data not shown) by comparing MCAO with sham-operated animals at any time. Furthermore, by costaining coronal brain cryosections from Foxp3EGFP reporter mice with DAPI, we could find GFP-positive Tregs undergoing mitosis in the infarct area (Supplementary Figure 2). These results suggest that a proliferation of Tregs may occur locally in the ischemic hemisphere between days 7 and 14 after MCAO.

Figure 5.

Figure 5

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(A, B) Ki-67 expression in Foxp3+ regulatory T cells (Tregs) after middle cerebral artery occlusion (MCAO). (A) Proportion of Ki-67+ cells in Foxp3+ Tregs in ischemic hemisphere compared with cervical lymph nodes (LN) and inguinal LN. (B) Proportion of Ki-67+ cells in Foxp3+ Tregs in cervical LN and inguinal LN compared with sham-operated animals on day 14. *P<0.05; **P<0.01; ***P<0.001. Data are presented as mean±s.d. and n=4 to 12 per group. (C) Expression of interleukin-2 (IL-2) mRNA in the ischemic hemisphere analyzed with a TaqMan Assay. n.d.=not detectable. Data are presented as mean±s.d. (D) Foxp3+ frequencies after transfer of naive CD4+/CD45RBhigh cells depleted of Foxp3+ cells compared with sham-operated animals 14 days after MCAO. Data are presented as mean±s.d. and n=5 to 7 per group.

Interleukin-2 has been described in the literature to be important for Tregs functioning, homeostasis, and proliferation.22, 23 We detected IL-2 mRNA being expressed on days 14 and 30 in the ischemic hemispheres. We could not detect any expression of IL-2 mRNA in contralateral hemispheres, in sham control, and in the ischemic hemispheres from day 7 (Figure 5C).

Middle Cerebral Artery Occlusion Does Not Lead to a De Novo Induction of Regulatory T Cells

We next asked if Tregs are de novo induced in the brain or periphery after MCAO. We transferred 5 × 106 naive CD4+/CD45RBhigh cells depleted of Foxp3EGFP+ Tregs together with 1 × 107 B cells into RAG1−/− mice 1 day before MCAO and analyzed the lymphatic organs, blood and brain hemispheres 14 days after MCAO by FACS. We found transferred CD4+ cells in the LN, spleen, and blood and an increased amount of CD4+ cells in the ischemic hemisphere of mice injected with naive CD4+/CD45RBhigh compared with contralesional hemisphere and sham controls 14 days after MCAO (data not shown). Indeed, we also identified a small percentage of de novo induced Foxp3+ Tregs in the brain and in the periphery, but no significant differences were seen compared with sham controls (Figure 5D). Thus, no specific MCAO driven de novo induction of Tregs could be detected.

In previous work it has been reported that RAG1−/− develop smaller infarct sizes due to the lack of T cells.3 In our study, no differences were seen after comparing MRI lesion volumes from RAG1−/− mice with C57BL/6J mice used in the flow cytometry experiment from Figure 1 (Supplementary Figure 3).

Delayed Depletion of CD25+ Regulatory T Cells Does Not Worsen Long-Term Outcome

To test the functionality of Tregs in the context of a late immune response, we injected a CD25+ Tregs depleting antibody on days 3 and 14 after MCAO. An overview of the different time points of MRI, gait analysis, and flow cytometry after MCAO is delineated in Figure 6A.

Figure 6.

Figure 6

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(A) Overview of experimental setup for middle cerebral artery occlusion (MCAO) with depletion of regulatory T cells (Tregs). (B) FACS analysis of blood cells and cells from ischemic hemispheres after treatment with anti-CD25 or isotype. The proportion of the CD25+ fraction in the green fluorescent protein (GFP+) Tregs subset was measured and the mean of the control group was set as 100%. Individual data points of both groups were calculated in relation to it. Data are presented as scatter dot plots with mean±s.d. and n=11 (anti-CD25) and n=9 (isotype). **P<0.01; ***P<0.001. (C) Exemplary magnetic resonance imaging (MRI) slice of a mouse treated with anti-CD25. On day 3 after MCAO, the lesion can be assessed with T2-weighted MRI by the means of water accumulation (shown in white) in the ischemic brain area. On day 27, defect and scarred tissue is seen in the lesioned area. Additionally, the ischemic hemisphere is atrophic and cerebral spinal fluid (in white) fills up part of the lost tissue.39 First the volume of damaged tissue was subtracted from the ipsilesional hemisphere. The lesion volume was determined indirectly by subtracting latter from the contralateral hemisphere. (D) MRI lesion volumes on days 3 and 27. Data are shown as box plots with interquartile, median, and range. No differences were seen between animals treated with anti-CD25 or isotype. (E) Relative percental MRI lesion volume (LV) of animals from day 27 was calculated in relation to day 3 after MCAO. Data are shown as box plots with interquartile, median, and range. No differences were seen between animals treated with anti-CD25 or isotype.

With flow cytometry, we detected CD25+ Tregs being reduced in the blood by 74% compared with the isotype control group 9 days after MCAO. On day 30, we could see a reduction of CD25+ Tregs by 36% in the blood and by 20% in the ischemic hemisphere (Figure 6B). Furthermore, we observed on day 30 a reduction of Foxp3−/CD25+ cells in cervical LN and spleen from mice treated with CD25 antibody, but no changes were seen in ischemic hemispheres. In blood samples from days 9 and 30, only minor amounts of Foxp3−/CD25+ cells were detected in both groups (Supplementary Figure 4).

Magnetic resonance imaging was used to determine lesion volumes on day 3 and day 27 (Figure 6C). On day 3, the median lesion volume was 37 mm3 in the group receiving anti-CD25 treatment and 38 mm3 in the control group. On day 27, the calculated median of the lesion volume was 44 mm3 in the anti-CD25 group and 36 mm3 in the control group. Statistically no significant differences were detected between day 3 and day 27 and comparing both treatment groups on day 27 (Figure 6D). To correct for the variance introduced by the MCAO preparation, we calculated the relative lesion volume by dividing the final lesion volume at day 27 by the initial lesion volume. In the anti-CD25 group, the median of the relative lesion volume was 117.0% and in the control group 110.2%. No significant difference was detected between both groups (Figure 6E).

A gait analysis was performed on days 14 and 27 after MCAO. Investigated gait parameters that are affected by MCAO are described elsewhere in detail.16 Here, we present a part of the gait parameters that are altered due to MCAO. Phase dispersion is a valuable parameter to assess interpaw coordination. Comparing phase dispersion from front left to right hind paw mice showed a less coordinated walk 14 days after MCAO compared with baseline (Figure 7A). However, no significant differences were seen directly comparing anti-CD25-treated animals and control. Similar results, concerning coordination, were seen in the regularity index of step sequence (Figure 7B). Comparing the maximal contact area of the right hind limb before and 14 or 27 days after MCAO respectively, we observed a clear decrease and animals showed less use of the right hind paw after MCAO, as indicated by duty cycle, but no changes took place due to CD25+ Tregs depletion (Figures 7C and 7D). Additionally, the normalized swing speed was lower after MCAO and the stand time in the right front limp was increased, presumably compensating the less effective use of the hind limbs. In both parameters, no differences occurred after depletion of CD25+ Tregs (Figures 7E and 7F).

Figure 7.

Figure 7

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Gait parameters 14 and 27 days after middle cerebral artery occlusion (MCAO) with comparison of anti-CD25 (−) and isotype (+) treated animals. (A) Comparison of the phase dispersion from left front to right hind paw (LF−>RH) of animals treated with anti-CD25 (−) or isotype (+). (B) Regularity index; (C) maximal contact area of the right hind paw (RH); (D) duty cycle of RH; (E) normalized swing speed of RH; (F) stand time of right front paw (RF). (A–F) Data are presented as box plots with 5 to 95 percentile whiskers. Values before stroke are represented in the background as gray boxes, in parallel to the box plots in darker gray the first to third quartile and in light gray the 5 to 95 percentile. The dotted line indicates the median. Comparison of effect sizes; n=20.

On day 30, an accumulation of CD45dim/CD11b+ microglia, CD45high/CD11b+ cells and CD4+ cells occurred in the ischemic hemisphere and an elevated proportion of microglia were MHCII+ in both groups. Though, no differences were detected in the amount of accumulated immune cells and MHCII+ expressing microglia between animals treated with anti-CD25 and control (Supplementary Figure 5).

Discussion

An ischemic infarct is accompanied by early activation of innate immune cascades, during which the blood–brain barrier is compromised and leukocytes infiltrate into the brain.1, 2, 5 Although studies have shown different subsets of immune cells to accumulate in the ischemic hemisphere within hours, overall little is known about the long-term modulation and activation of the immune system following stroke. An influx of T lymphocytes and an accumulation of microglia, macrophages, and DCs in the ischemic hemisphere within 1 week have been described previously.1, 2, 24

In the here present study, we discovered a second increase of CD4+ cells in the ischemic hemisphere that was clearly distinct from the first, widely accepted wave of lymphocytes that takes place shortly after stroke. This second infiltration peaked around day 14 and persisted until day 30 after stroke (Figures 1B and 1C). The appearance of a very moderate number of CD4+ cells in the brain at day 7 foreshadows this second immune response in the brain. Using the two independent approaches of flow cytometry and immunohistochemistry, we also identified a late accumulation of Foxp3+ Tregs after MCAO (Figures 1C and 1D and 2C).

Immature DCs are known to induce or reactivate Tregs in the periphery,25, 26, 27 and microglia, the brain's immune competent cells, can become MHCII+ after activation.28 Dendritic cells and macrophages are also known to invade brain parenchyma after brain injury. This led us to analyze the distribution of all three cell types in one study framework. While we detected macrophages and DCs on each analyzed day (Figures 3B and 3C), the overall and MHCII+ number of microglia was higher at days 14 and 30 in the ischemic hemisphere and this increase occurred in parallel with the delayed accumulation of CD4+ cells and Tregs (Figures 3B and 3C). In the autoimmune disease model, EAE microglia play a variety of roles in activating or regulating immune response depending on the phase of disease.29 It could be that microglia are related to the maintenance of Tregs at late time points after MCAO.

In this study, we investigated whether the prolonged accumulation is due to the proliferation of Tregs, using the Ki-67 protein as a marker for proliferation.30, 31 Regulatory T cells can proliferate after an antigenic stimulation, which could explain why Tregs accumulate in the brain late after stroke.32, 33 Interestingly, we found that around 60% of Tregs were in a proliferative state at day 7, keeping in mind that only a small number of CD4+ cells were found. Additionally, on day 14 we saw a strong increase of Tregs, of which again around 60% were positive for the Ki-67 protein in the ischemic hemisphere. The level of Ki-67+ Treg on these days was about threefold higher than in lymphatic organs and blood on any analyzed day and than in the ischemic hemispheres from day 30 (Figure 5A). One possibility is that activated Tregs enter a continuous proliferative state in the periphery and remain in this state during and after infiltration into the brain, since clonal expansion can proceed in the absence of further antigen exposure.34 We could not see any changes in Ki-67 expression in the periphery and, since we observed a direct contact between Tregs and MHCII expressing cells in the ischemic hemisphere, it is possible that infiltrated Tregs are reactivated after infiltrating into the central nervous system. In this context, we detected an elevated expression of IL-2 mRNA on days 14 and 30 (Figure 5C). Interleukin-2 is known to be important for controlling proliferation of Tregs and could be related to Tregs proliferation in the brain late after MCAO.22, 23 Indeed, we could not detect any expression of IL-2 mRNA on day 7 when a high level of Tregs was Ki-67+. This could be due to the low amount of CD4+ cells found at day 7 since we analyzed the expression of IL-2 mRNA in whole hemispheres.

The high level of Ki-67+ Tregs leads us to speculate that MHCII+ microglia form a micromilieu that contributes to the activation or reactivation of invading Tregs. While we cannot exclude that invading DCs or macrophages also contribute to Treg activation processes, the amount of microglia found in the ischemic hemisphere was 10- to 40-fold higher than that of macrophages or DCs. Moreover, at days 14 and 30 after MCAO, the MHCII+ microglia were highly present in the ischemic hemisphere (Figures 3B and 3C). In addition, our immunohistological experiments showed predominantly Iba1-positive cells directly in the infarct area (Figures 4A and 4B).

To analyze whether a de novo induction of Tregs takes place in the brain or periphery following ischemic stroke, we used an established method, namely, adoptive transfer of naive CD4+CD45RBhigh cells into T-cell-deficient mice.35 The induction of Tregs from naive CD4+ cells 14 days after MCAO was not significantly altered in either lymphatic organs or brain. Indeed, we saw an accumulation of CD4+ cells in the brain 14 days after the ischemic insult, suggesting an activation of peripheral immune cells in this setup. It could be that RAG1−/− mice are lacking components of the immune system, which are required to form a molecular micromilieu necessary for a de novo generation of Tregs in the brain after MCAO. As a matter of fact, the RAG1−/− mouse lacks immune cells such as memory T cells and CD8+ cells in this setup.

In a further experiment we wanted to clarify the function of CD25+ Tregs in the late phase after MCAO. We started with the depletion protocol 3 days after MCAO and repeated the antibody injection on day 14 to guarantee sufficient depletion of CD25+ Tregs at the time a strong accumulation of CD4+ T cells and Foxp3+ Tregs occurs in the ischemic hemisphere. To assess the neurological outcome we did measurements of the lesion size by MRI and an analysis of gait parameters that are affected by MCAO (Figure 6A). A strong depletion of CD25+ Tregs was detectable in the blood on day 9 and was still significant on day 30 in the blood and in the brain, but no effect was seen regarding activation of microglia and accumulation of immune cells in the ischemic hemispheres on day 30. In a previous study, administration of the CD25 antibody lead to depletion of activated CD4+ cells.36 In our present study, we could see a reduction of Foxp3−/CD25+ cells characterized as activated CD4+ cells in cervical LN and spleen on day 30 after MCAO. Despite the observation that no changes regarding activated CD4+ cells occurred in the ischemic brain, this observation should be taken into account when using CD25 antibody for Tregs depletion. Nevertheless, we could not detect any changes related to the neurological outcome in animals depleted of CD25+ Tregs. A reason for the negligible effect of CD25+ Tregs on the neurologic outcome could be related to the infarct size. The effect of CD25+ Tregs was detectable in models with small lesion sizes of around 15 mm3, but not in mice with lesion sizes >100 mm3.10 In another study where Foxp3+ Tregs were depleted in the acute phase of MCAO and infarct sizes were around 50 mm3 neither any effects on infarct size nor functional outcome were detected.37 We believe that this is an important point, because specific cell types of the immune system might be differentially relevant in different models of stroke. This must be taken into account when translating results into clinical practice. Another point could be the start of the depletion protocol at day 3 (Figure 6A), which differed from previous published work. It could be that in the late phase the function of Tregs varies and is associated with repair mechanisms such as scar formation, tissue remodeling, or revascularization.38 Taken together, this experiment shows that in this specific model for ischemic stroke, CD25+ Tregs are not relevant in limiting detrimental inflammatory processes that affect lesion size and functional improvement in the late phase after MCAO.

Our present study detected for the first time the occurrence of an immunologic event temporally separate from the first infiltration of immune cells, beginning within hours after an ischemic insult. The late accumulation of CD4+ T cells and antigen presenters in the ischemic brain might be of significance when targeting the immune system for therapeutic purposes after stroke.

Acknowledgments

The authors thank Dr Ulrike Steckelings and Kristin Lucht for supporting us with the MCAO surgery. The authors also thank Susanne Mueller for the support with the MRI studies.

The authors declare no conflict of interest.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

This work was supported by the SFB-TRR 43, Berlin-Göttingen 2008–2011 (project B4) and 2012–2015 (project B5).

Supplementary Material

Supplementary Information
Click here for additional data file. (2.3MB, pdf)

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