Visualizing the activation of encephalitogenic T cells in the ileal lamina propria by in vivo two-photon imaging

C57BL/6J wild-type mice, TCR^MOG mice (also known as 2D2) (10), OT-II mice (11), SMARTA mice (12) and 2D2 × TH PhAM^T mice (crossing PhAM^floxed reporter mice (13), CD4-Cre mice (14) and 2D2 mice and BCRMOG mice (also known as TH) (15) were bred in the Core Facility Animal Models of the Biomedical Center, LMU Munich, or a specific pathogen-free (SPF) facility at the Technical University of Munich. 2D2 mice have been reported to develop spontaneous EAE in around 4% of cases; however, the rate at which mice get spontaneous EAE in our facility is 0%. The GF mice were bred in the animal facilities of the Technical University of Munich (School of Life Sciences Weihenstephan) and were kindly provided by Dirk Haller. The local authority (Regierung von Oberbayern) approved all mouse experiments.

Lymph nodes and spleens were passed through a 40-μm cell strainer (Greiner Bio-One), followed by gravity centrifugation (400 g, 4 °C, 5 min). Blood was drawn from the heart into a syringe that had been flushed with heparin (5,000 U/mL) (Sigma-Aldrich) in PBS. Spleen and blood samples underwent erythrocyte lysis with ACK solution [NH₄Cl 8.024 mg/l (Merck), KHCO₃ 1.001 mg/l (Merck), and EDTA.Na₂·2H₂O 3.722 mg/l (Merck)].

For retroviral T cell transduction experiments, B cells from lymph nodes and spleens were first depleted from the cell suspensions using the Dynabeads B220 depletion kit (Thermo Fisher). The remaining T cells were then cultured in the presence of 0.5 μg/mL anti-CD3 (500A2, Invitrogen) and anti-CD28 (37.51, Invitrogen)-stimulating antibodies in complete DMEM (Sigma-Aldrich) [supplemented with 2 mM L-Glutamine (Sigma-Aldrich), 100 μg/mL Penicillin/Streptomycin (Sigma-Aldrich), 36 mg/l Asparagine (Sigma-Aldrich), 1 mM Sodium-Pyruvate (Sigma-Aldrich), 10 mL/l Nonessential amino acids (Sigma-Aldrich), 4 μL/l β-Mercaptoethanol (Merck KGaA), and 10% fetal calf serum (Sigma-Aldrich)] at a concentration of 2 × 10⁶ cells/ml in a 12-well tissue culture plate (2 mL/well) for 2 d.

For the generation of retrovirus harboring Twitch-2B DNA, HEK293T cells (American Type Culture Collection) were cultured in complete DMEM (see above) and transfected using the polyethylenimine (PEI max) (Polysciences) transfection method with 6 μg of pMSCV-Δneo-Twitch-2B and 3.5 μg of pCL-Eco plasmid. The culture medium was refreshed at 24 h after PEI transfection. The virus-containing supernatant was harvested after 48 h and 72 h, and viral particles were concentrated using Amicon® Ultra Filters (100-kDa cutoff, Merck).

After 2 d of culture, T cells were resuspended at a concentration of 4 × 10⁶ cells/mL in complete DMEM with the concentrated retroviral supernatant at a dilution of 1:10, supplemented with 8 μg/mL polybrene (Sigma-Aldrich) and 10 ng/mL IL-2 (PeproTech). The T cells were spin-infected in 12-well plates with 0.5 mL per well (2,000 g, room temperature, 90 min). Typically, transduction yield was around 50%.

A total of 20 to 30 × 10⁶ Twitch-2B transduced T cells were transferred intravenously into C57BL/6J wild-type SPF or GF mice 1 d after retroviral transduction.

Prior to surgery, the mice were anesthetized with a cocktail of medetomidine, midazolam, and fentanyl [MMF; (5, 0.5, and 0.05 mg per kg body weight, respectively], tracheotomized, and ventilated with 1.5% isoflurane throughout the surgery and imaging procedure (16). Their body temperature was regulated using a custom-made heated microscope stage, and physiological parameters were constantly monitored during imaging. The blood vessels were visualized by i.v. infusion of Texas Red-conjugated dextran (50 μg, 70 kDa, Invitrogen). For one set of experiments, 100 µg of an anti-MHCII blocking antibody (clone M5/114.115.2, BioLegend) was injected (i.v.). During imaging, the abdomen was opened by midline incision. The ileum, more specifically close to the cecum, was taken out as little as necessary, and a PP-rich section was placed on a custom-made stage padded by dampened tissues. The intestinal tube was opened with a longitudinal incision of approximately 1 cm using a cautery pen (Bovie). Both sides were folded back onto the dampened tissues on the stage to have a flat layer exposing the ileal LP. The intestinal content was carefully removed before fixing the gut with a cover slip and tissue adhesive (3M Animal Care Products). The cover slip was pressed down so that peristaltic movements could not disturb image acquisition (17).

Photoconversion was performed as previously described (16) with some modifications. Briefly, asymptomatic 4-wk old 2D2 × TH PhAM^T mice were anesthetized by i.p. injection of medetomidine (0.5 mg/kg), midazolam (5 mg/kg), and fentanyl (0.05 mg/kg). The fur was removed from the abdominal area using hair removal cream (Veet), and the body temperature was regulated by placing the mice on a custom-made heat mat. To expose the ileum, a 2-cm incision on the skin and the abdominal wall was made first. From the incision, the cecum, followed by the ileum, was pulled on a compression bandage soaked in 0.9% sodium chloride solution. T cells in the ileum were photoconverted from green to red fluorescence by irradiating the exposed ileum for 10 min with a 405-nm diode laser (LuxX 405-60, Soliton, OMI.115.15001). After the irradiation, buprenorphine (0.05 mg/kg) was given s.c. for analgesia, and the incisions were closed by sutures. The anesthesia was antagonized by i.p. injection of atipamezole (2 mg/kg), flumazenil (0.5 mg/kg), and naloxone (1.5 mg/kg). Mice were killed for analysis 3 d after the irradiation.

For imaging of the ileal PP, the same procedure as for the ileal LP imaging was applied with minor modifications. For the PP imaging, the intestinal tube was not opened, but instead, the PPs were imaged from the outside. Therefore, the ileum with a PP facing the top was placed on the custom-made stage padded by dampened tissues and fixed from the top with a cover slip.

Time-lapse two-photon laser-scanning microscopy was performed using a SP2 or SP8 MP microscope (Leica) as described previously (17). The SP2 system was equipped with a 10 W Millenia/Tsunami laser (Spectra Physics, Darmstadt, Germany). The excitation wavelength was tuned to 835 nm and routed through a water immersion objective (25×, NA 0.95, Leica). With a zoom factor of two, areas of 240 × 240 μm size were scanned, and 30- to 50-μm z-stacks were acquired with 3- to 4-μm z-step size. The acquisition rate was 25-s interval time with images line-averaged twice. Fluorescent signals were detected using nondescanned photomultiplier tube detectors equipped with 475/50-nm, 537/26-nm, 630/69-nm, and 685/40-nm bandpass filters (Semrock).

Multiphoton microscopy using the SP8 MP microscope was performed at the Core Facility Bioimaging of the Biomedical Center, LMU Munich. The SP8 system featured a pulsed InSight DS+ laser (Spectra Physics), and emission was tuned to 841 nm and routed through a water immersion objective (25×, NA 1.0, Leica). Using a 2× zoom, areas of 221 × 221 μm were scanned and acquired in 30- to 50-μm z-stacks with 3- to 4-μm step size and an acquisition rate of 25 s per z stack. Images were recorded with external, nondescanned hybrid photodetectors (HyDs) equipped with 483/32-nm, 535/30-nm, 585/40-nm, and 650/50-nm bandpass filters.

Time-lapse images were acquired with Leica LCS software (Leica) for the SP2 system and with LAS X software (Leica) for the SP8 setup, respectively, and processed and analyzed using ImageJ (NIH, Bethesda, MD, USA) as described previously (17).

A Gaussian blur filter was used before Z-projecting the stacks with maximum intensity to generate two-dimensional movies. For Twitch-2B-transduced cells, ratiometric pseudocolor images were obtained by dividing the cpVenusCD (FRET) by the mCerulean3 (CFP) channel and changing to a fire lookup table. To analyze the calcium ratios, the cell shapes were manually outlined in each time frame in the 2D maximum projection to create a region of interest (ROI) for every cell to be tracked. The bleed-through of CFP into the YFP channel was determined to be 44%; hence, the FRET signal was corrected as YFP = FRET – 0.44 × CFP. The average signal intensities of all pixels in each ROI were defined to calculate the calcium ratio at every given time point.

In order to obtain efferent mesenteric lymphatic fluid and thus lymphocytes exiting the gut, the efferent mesenteric lymph duct was cannulated. Thirty min prior to cannulation, the animals were orally gavaged with 200 μL olive oil to visualize lymphatic vessels. The cannulation technique was modified from that of Druzd et al (18). In brief, the mice were anesthetized with MMF, and the skin was incised along the left costal arch, and the peritoneal cavity was opened. The efferent mesenteric lymphatic duct locates below the left kidney. The cannulation tube [29G needle (BD) inserted into the tip of a 0.61-mm polyethylene tube (Warner Instruments)] was flushed with 15 μL heparin (5,000 U/mL). The cannulation tube was carefully inserted into the lymph duct, and the fluid was collected in a 29G syringe (BD). The liquid was washed with 10 ml PBS prior to flow cytometric staining.

Cell suspensions from spleen, blood, and efferent mesenteric lymphatic fluid were stained with the following fluorochrome-conjugated antibodies: APC anti-mouse CD3 (clone 17A2, BioLegend, 1:200), FITC anti-mouse CD4 (RM4-5, BioLegend, 1:200), PE anti-mouse CD8 (53-6.7, BioLegend, 1:200), PerCP/Cy5.5 anti-mouse CD19 (6D5, BioLegend, 1:200). For dead cell exclusion, cells were stained with SYTOX^TM Blue Dead Cell Stain (Invitrogen), and to avoid Fc-receptor binding, anti-mouse TruStain FcX (BioLegend) was used.

After the staining, CD4⁺ T cells were sorted on a FACSAria IIIu (BD) using a 70-μm nozzle. Cell sorting was performed at the Core Facility Flow Cytometry of the Biomedical Center, LMU Munich. Flow cytometry data were analyzed using FlowJo (Tree Star Inc.).

For the analysis of photoconverted T cells, single-cell suspensions from the spleen and mononuclear cells from the CNS were prepared as previously described for flow cytometry (16). The following antibodies were used: APC anti-mouse CD62L (MEL-14, BD, 1:400), BV421 anti-mouse CD44 (IM7, BD, 1:400), and BV786 anti-mouse CD4 (RM4-5, BD, 1:800). Dead cells were detected with a near-infrared LIVE/DEAD dye (Thermo Fisher, 1:500), and nonspecific antibody binding to FC receptors was blocked with anti-mouse CD16/CD32 (2.4G2, BD; 1:200). The samples were acquired on a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software (10.8.0).

Total RNA was isolated from sorted CD4⁺ T cells using the RNAeasy Plus microkit (Qiagen) as described previously (17). Quality and integrity of RNA was controlled on a Bioanalyzer 2100 (Agilent Technologies). Transcriptome analysis and subsequent bioinformatics were performed by BGI Group using their DNBseq™ platform. The sequence alignment to the reference genome was performed using HISAT2 (Hierarchical Indexing for Spliced Alignment of Transcripts two) (19). Differentially expressed genes (DEGs) were detected using DEseq2 (20).

The statistical evaluation was performed using Prism software (GraphPad, San Diego, CA) as described in the figure legends. Significance was indicated by the P-value as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

We focused on the activation events taking place in the ileal wall containing microenvironments favoring immune cell activation. To calibrate calcium signaling in situ, we started out by imaging polyclonal CD4⁺ T cells which were transduced to express the calcium indicator Twitch-2B at a high proportion prior to transfer into recipient mice (Fig. 1 A and B). Three days after transfer, Twitch-2B-labeled polyclonal CD4⁺ T cells moved through the LP occasionally showing increased intracellular calcium (Fig. 1 C–E and Movie S1). We quantified the relative intracellular calcium levels (shown as the ratio of YFP/CFP) and instantaneous velocity at each time point of each cell (Fig. 1C). As observed previously in rat T cells (9), calcium signaling and cellular velocity were negatively correlated (Fig. 1D). The threshold to classify calcium ratios as calcium signaling was set to be above 95% of all calcium ratios identified in polyclonal T cells.

As the next step, we used our protocol to transduce MOG-specific encephalitogenic CD4⁺ T cells from 2D2-transgenic mice. After retroviral gene transfer, Twitch-2B-expressing MOG-specific CD4⁺ T cells were transferred into recipient mice for intravital imaging (Movie S2). In MOG-specific 2D2 CD4⁺ T cells, the frequency of calcium signaling was significantly higher than in polyclonal T cells (Fig. 2 A and B) with a negative correlation with velocity similar as in polyclonal T cells (Fig. 1C). The increased calcium signaling coincided with a decrease in the average velocity per cell (6.4 µm/min in polyclonal vs 4.8 µm/min in MOG-specific CD4⁺ T cells) (Fig. 2C). Not only the frequency but also the duration of calcium signaling was increased in MOG-specific vs. polyclonal T cells. Both the duration and frequency of long-term calcium signaling (longer than 2 min) were significantly increased in MOG-specific vs. polyclonal CD4⁺ T cells with a maximum duration of 16.4 vs. 4.62 min and average duration of 1.5 vs. 0.86 min. Among the MOG-specific CD4⁺ T cells, 23.5% exhibited long-term calcium signaling as compared to only 3.7% in polyclonal CD4⁺ T cells (Fig. 2 D and E). In contrast, short-lasting signals (shorter than 2 min) were observed at similar rates in both polyclonal and MOG-specific T cells (Fig. 2 D and E).

In order to understand whether the stimulation of MOG-specific CD4⁺ T cells was restricted to the LP or could also be detected in the PPs, we performed intravital microscopy of Twitch-2B-transduced CD4⁺ T cells in those lymphoid follicles on the ileal surface. As in the LP, polyclonal CD4⁺ T cells served to define the threshold in PPs. The timeline of intravital microscopy in PPs was the same as in the LP shown in Fig. 1A. Representative time-lapse pictures displayed the motility and calcium activities of polyclonal CD4⁺ T cells in a PP (Fig. 3A). The analysis of the calcium ratios in the polyclonal CD4⁺ T cells revealed that the background signal in the PPs was a bit higher than in the LP, so the threshold was set to 0.73 (95% of the calcium ratios were below that value) (Fig. 3C). The subsequent examination of MOG-specific 2D2 CD4⁺ T cells failed to detect increased calcium levels with 5.8% above threshold (Fig. 3 B and D). In contrast to the stimulation in the ileal LP, the proportion of T cells with long-term calcium signaling in the PP was not changed significantly depending on the TCR specificity of the cells. Likewise, the fraction of cells with short-term high-calcium signaling was not significantly increased in MOG-specific 2D2 CD4⁺ T cells over polyclonal CD4⁺ T cells [40.4 ± 7.8% (T_MOG cells) vs. 34.2 ± 8.2% (polyclonal T cells)] (Fig. 3E). The longest calcium signaling lasted 20 min in a MOG-specific CD4⁺ T cell and 13 min in a polyclonal CD4⁺ T cell, and in both, most calcium signals were lasting less than 2 min (Fig. 3F).

In order to control for endogenous MOG or its mimes as activators of 2D2 CD4⁺ T cells, two transgenic T cell lines responding to antigens distinct from MOG, OVA-specific CD4⁺ T cells from OTII-transgenic mice and LCMV-specific CD4⁺ T cells from SMARTA mice were used. Dietary contamination of OVA in the mouse chow and inadvertent infection with LCMV were ruled out. Unexpectedly, OTII CD4⁺ T cells showed similar calcium activities as 2D2 CD4⁺ T cells (Fig. 4 A and C and Movie S3). Also, SMARTA CD4⁺ T cells showed an increased amount of calcium signals above threshold similar to 2D2 and OTII CD4⁺ T cells (Fig. 4 B and D). The maximum durations of the calcium spikes were 13 and 8.4 min for OVA-specific CD4⁺ T cells and LCMV-specific CD4⁺ T cells, respectively, with an average duration of 1.2 min in OVA- and 1.1 min in LCMV-specific CD4⁺ T cells (Fig. 4E). The proportion of T cells exhibiting long-term calcium signaling was increased in all TCR-transgenic T cells as compared to polyclonal CD4⁺ T cells, whereby the increase was less pronounced in the LCMV-specific CD4⁺ T cells [4.2 ± 1.9% (polyclonal) vs. 22.8 ± 9.2% (MOG) vs. 26 ± 17.2% (OVA) vs. 12.7 ± 2.2% (LCMV)]. Not only the long-term spikes but also the proportion of T cells with short-term calcium signaling was higher in the TCR-transgenic cells: 31.6% of polyclonal CD4⁺ T cells exhibited short-lasting high-calcium signals in contrast to 52.7% in MOG-specific CD4⁺ T cells, 63.6% in OVA-specific CD4⁺ T cells and 58.3% in LCMV-specific CD4⁺ T cells (Fig. 4F).

MOG TCR–transgenic mice develop spontaneous EAE under SPF but not under GF conditions, indicating that the encephalitogenic T cells are activated via microbiota or derived factors from them (3). To test this, we transferred Twitch-2B-expressing 2D2 CD4⁺ T cells into GF C57BL/6J mice. In the GF ileal LP of recipient mice, the proportion of calcium ratios above threshold was drastically reduced in MOG-specific CD4⁺ T cells as compared to the LP of recipients housed under SPF conditions (Fig. 5A and Movie S4). The vast majority of T cells (70.5 ± 5.4%) traveled through the GF ileal LP spontaneously without showing any calcium activities (Fig. 5C). Occasional short-term calcium signals were observed in the rest of the MOG-specific CD4⁺ T cells (29.5 ± 5.4%), whereas long-term calcium signaling was completely missing in the absence of gut microbiota (Fig. 5 B and C). These results indicate that microbiota play a pivotal role in the induction of calcium signaling and hence activation of T cells in the ileal LP.

These experiments were complemented by applying anti-MHC class II blocking antibodies i.v. during intravital imaging. As shown in Fig. 5D, the intravenous injection of anti-MHC class II antibodies diminished calcium signaling in MOG-specific CD4⁺ T cells significantly. The blocking antibody reduced the maximum time of calcium signaling from 18 min to 2.5 min (Fig. 5E), led to a decrease in the frequency of short-term calcium signaling, and subsequently suppressed long-term calcium signaling (Fig. 5F).

We next evaluated the effect of exogenous antigens. Twitch-2B-transduced OVA-specific CD4⁺ OT-II T cells were adoptively transferred 3 d prior to microscopy along with an oral bolus of 4 mg OVA protein together with Texas-red conjugated 70-kDa dextran shortly before starting intravital microscopy. The fraction of elevated calcium ratios rose significantly over the calcium ratios of OVA-specific CD4⁺ T cells without the exogenously applied antigen (Fig. 6A, compare Fig. 4A) (22% with vs. 14% without antigen). The intense red fluorescent signal indicated the penetration of orally applied antigen into the ileal LP at the time of microscopy (Fig. 6B). Interestingly, the calcium response of the stimulated CD4⁺ T cells was time dependent: Most of the calcium signaling in the early phase was long lasting and then decreased over time to be replaced after 77 min completely by short-lasting calcium signaling. This suggests that the exogenous OVA entered the small intestine quickly and was promptly processed to transiently enhance T cell stimulation (Fig. 6C).

Calcium signaling triggers phenotypical changes in T cells. These should be demonstrable in CD4⁺ T cells emigrating from GALT. To this end, the efferent lymphatic vessels were cannulated, and lymphocytes were collected from the lymphatic fluid (18). The numbers of lymphocytes recovered from the GALT efferent lymphatic vessels differed depending on the mouse housing condition and genetic background (Fig. 7A). The highest counts were noted in the lymphatic fluid from mice housed under SPF conditions; among these, wild-type mice yielded a higher cell concentration (477 ± 313 cells/µL) than mice with a MOG-specific TCR transgene (237 ± 109 cells/µL). GF mice of either genetic background provided lymphatic fluid with further reduced cell numbers [205 ± 330 cells/µL (wild-type (wt) mice) and 101 ± 102 cells/µL (GF mice with MOG TCR)].

In the efferent mesenteric lymphatic fluid of wt mice, CD4⁺ T cells constituted the predominant lymphocyte subset independent of the animals’ hygiene condition [47% (SPF) vs. 48% (GF)]. In MOG TCR–transgenic mice, the CD4⁺ population was increased to 66% and 51% in SPF and GF mice, respectively. In contrast, CD8⁺ T cells, which were almost absent in the mesenteric lymphatic fluid [2% (SPF), 0.1% (GF)], which is probably due to the genetic modification by TCR transgene (10) (Fig. 7B).

By taking advantage of a photoconvertible protein, we tracked the migration of MOG-specific CD4⁺ T cells from the ileal LP. Three days after photoconversion in the ileum, around 4% of CD4⁺ T cells in the CNS harbored the photoconverted protein (Fig. 7C), suggesting that these T cells had freshly migrated to the CNS from the ileum. Further phenotypical analysis of the T cells showed that the photoconverted T cells in the CNS contained more antigen-experienced T cells (CD44^high, CD62L^low) than naive T cells, whereas the photoconverted T cells in the spleen contained more naive T cells (CD44^low, CD62L^high) than antigen-experienced T cells (Fig. 7D). These results indicate that antigen-experienced T cells migrated from the ileum into the CNS prior to clinical EAE.

CD4⁺ T cells were sorted from lymphatic fluid, spleen, and blood as shown in the representative flow cytometry plots in SI Appendix, Fig. S1A, and bulk RNAseq was performed. The cluster dendrogram in SI Appendix, Fig. S1B shows that the MOG-specific CD4⁺ T cells have specific transcriptome profiles dependent on the tissue compartment and hygiene conditions as expected. The impact of the housing condition appeared to be bigger than the one depending on the location since most of samples from SPF and GF clustered separately (SI Appendix, Fig. S1B). A volcano plot shows DEGs in MOG-specific T cells between SPF and GF recipients (Fig. 7C). To get a deeper insight into the role of the gut microbiota, the comparison between MOG-specific 2D2 T cells collected from lymphatic fluid and the spleen in SPF mice was included in the analysis in addition to the housing conditions (Fig. 7D). Among the up-regulated genes in the lymphatic fluid of SPF recipients, six genes (Il22, Ccr6, Ahrr, Il17re, Il1r1, and Il23r) were related to Th17 cells, which are considered a key player to induce CNS inflammation. Flow cytometric analysis confirmed an increased number of Th17 cells in 2D2 mice compared to wild-type mice, whereas the numbers of Th1 cells were similar (Fig. 7G). In addition, some genes, which are known to be involved in lymphocyte migration, were up regulated. For example, Ccr6 encodes a receptor for CCL20 and is involved in T cell migration into the CNS (21). Fndc7 and Lrfn encode fibronectins. Wagner et al. showed that fibronectin is up-regulated on the surface of activated T cells and plays a role in the proliferative capacity of those T cells (22). Although the numbers of DEG are limited, the results suggest that the MOG-specific T cells in SPF mice, where they show a higher rate of calcium signaling, tend to have increased numbers of Th17 cells. In contrast, the transcriptome analysis of polyclonal T cells from the efferent mesenteric lymphatic fluid from SPF vs. GF mice did not reveal differential gene expression in genes related to immune functions, which is in line with the absence of increased calcium signaling in polyclonal T cells (SI Appendix, Fig. S1C).

I.J.B., P.F., H.W., and N.K. designed research; I.J.B., P.F., K.F.L., and S.T. performed research; D.A., D.B., H.Y., and T.K. contributed new reagents/analytic tools; I.J.B., P.F., and K.F.L. analyzed data; and I.J.B., H.W., and N.K. wrote the paper.

The authors declare no competing interest.