T cell dynamics during induction of tolerance and suppression of experimental allergic encephalomyelitis

Abstract

The cell dynamics associated with induction of peripheral T cell tolerance remain largely undefined. In this study, an in vivo model was adapted to two-photon microscopy imaging, and T cell behavior was analyzed on tolerogen-induced modulation. FcγR-deficient (FcγR(-/-)) mice were unable to resist or alleviate experimental allergic encephalomyelitis when treated with Ig-myelin oligodendrocyte glycoprotein (MOG) tolerogen, an Ig carrying the MOG35-55 peptide. However, when FcγR(+/+) dendritic cells (DCs) are adoptively transferred into FcγR(-/-) mice, uptake and presentation of Ig-MOG occurs and the animals were able to overcome experimental allergic encephalomyelitis. We then fluorescently labeled FcγR(+/+) DCs and 2D2 MOG-specific TCR-transgenic T cells, transferred them into FcγR(-/-) mice, administered Ig-MOG, and analyzed both T cell-DC contact events and T cell motility. The results indicate that tolerance takes place in lymphoid organs, and surprisingly, the T cells do not become anergic but instead have a Th2 phenotype. The tolerant Th2 cells displayed reduced motility after tolerogen exposure similar to Th1 cells after immunization. However, the Th2 cells had higher migration speeds and took longer to exhibit changes in motility. Therefore, both Th1 immunity and Th2 tolerance alter T cell migration on Ag recognition, but the kinetics of this effect differ among the subsets.

Figure 1. Agg. Ig-MOG treatment modulates both active and passive EAE

In (A), groups of adult FcγR^−/− mice were given i.v. 6 × 10⁶ purified FcγR^+/+ DCs and 10 × 10⁶ naïve 2D2 T cells. The next day the mice were given i.p. 600 μg agg Ig-MOG (circles) or control Ig-W (squares). One day later the hosts were induced for active EAE with 300 μg of MOG peptide and monitored daily for signs of paralysis. In (B), groups of FcγR^−/− mice were given 6 × 10⁶ purified FcγR^+/+ DCs and induced for passive EAE by adoptive transfer of 10 × 10⁶ MOG-activated 2D2 TCR transgenic splenocytes. The next day the mice were treated i.p. with 600 μg agg Ig-MOG (circles) or control Ig-W backbone without MOG peptide (squares) and monitored for signs of paralysis for the duration of the experiment. Groups of mice that did not receive any FcγR^+/+ DCs (triangles) were included for comparison purposes. In (C), groups of adult FcγR^−/− mice were induced for active EAE with 300 μg of MOG peptide and 4 days later were transferred i.v. with 6 × 10⁶ purified FcγR^+/+ DCs. The next day the hosts were treated i.p. with 600 μg agg Ig-MOG (circles) or the control Ig-W (squares). The mice were then monitored for reduction in disease severity for the duration of the experiment. Groups of mice that did not receive any FcγR^+/+ DCs (triangles) were included for comparison purposes. Each point represents the mean clinical score of 6–8 mice and is representative of 4 independent experiments.

Figure 2. Assessment of T cell and DC trafficking upon induction of tolerance with agg Ig-MOG

FcγR^−/− mice were given i.v. 6 × 10⁶ SNARF-labeled FcγR^+/+ DCs followed 6h later by 10 × 10⁶ CFSE-labeled naïve 2D2 TCR transgenic CD4 T cells. The next day the mice received i.p. 600 μg agg Ig-MOG or Ig-W and 36h later specimens from the liver, lung, intestine, spleen and lymph nodes were harvested. Cryosections were then prepared and examined under UV microscope. Images acquired in red and green channels were overlaid and analyzed for pattern of cellular migration. The data is representative of 3 independent experiments.

Figure 3. Analysis of T cell-APC contacts by fluorescence microscopy

FcγR^−/− mice were given i.v. 6 × 10⁶ SNARF-labeled FcγR^+/+ DCs followed 6 hours later by 10 × 10⁶ CFSE-labeled naïve 2D2 TCR transgenic CD4 T cells. The next day, the mice received i.p. 600 μg agg Ig- MOG. Cryosections were examined for T cell-DC contact under a UV microscope. Images were acquired in red and green channels and overlaid for analysis of the proximity of red DCs in conjunction with green CD4 T cells. Such proximity events were quantified manually. Untreated mice were included as control. A, Histology images are shown for a section from the inguinal lymph node at 36h to illustrate T cell-APC contacts when the mice are given agg Ig-MOG (lower panel) versus no antigen (upper panel). B, At 12, 36, 60, and 96h after antigen exposure a group of mice was sacrificed and specimens from spleen (sp), cervical (cx), axillary (ax) and inguinal (in) lymph nodes were collected as indicated in the pictogram at the top of the figure. Cryosections prepared from these organs were examined for T cell-DC contact as described above from untreated (control) or agg Ig-MOG (Ig-MOG) recipient mice. Each point represents the number of contacts ± SD of two different experiments. **p values are below 0.05 as analyzed by unpaired, Student t test.

Figure 4. In vivo imaging analysis of tolerized T cells

In (A) FcγR^−/− mice were adoptively transferred with 10 × 10⁶ CFSE-labeled 2D2 CD4⁺ T cells and 6 × 10⁶ FcγR^+/+ DC. The next day the hosts were given i.p. 600 μg agg Ig-MOG tolerogen (TOL) in saline or saline without Ig-MOG (NIL). The axillary and inguinal LNs were harvested at 48h post tolerogen injection and subjected to 2-photon imaging. The behaviors of the 2D2 T cells were analyzed in detail using the Imaris software program (Bitplane). 2-dimensional vectors of T cell movement are depicted with normalization of starting point to center of an XY grid (top panels) of images showing cell tracks (bottom panels). In (B) the graphs illustrate the velocity and motility coefficient of live cells with movement tracks of 8 frames or higher. The scatter plots (top panel) show representative lymph nodes for each condition. The bar graphs (bottom panel) show the cumulative events collected from the lymph nodes of 3 mice per condition. The line represents the mean of analyzed cells. *p<0.05 in comparison to NIL mice as analyzed by unpaired, Student t-test.

Figure 5. Tolerized T cells express activation markers and do not undergo apoptosis

In (A) FcγR^−/− mice recipient of 6 × 10⁶ unlabeled FcγR^+/+ DC and 10 × 10⁶ SNARF- labeled 2D2 CD4 T cells were given PBS (Nil) or 600 μg agg Ig-MOG (tolerized) the next day. Forty-eight hours later, lymph node cells were stained with ZVAD-FITC. ZVAD binding was analyzed on SNARF⁺ cells. In (B) FcγR^−/− mice recipient of 6 × 10⁶ unlabeled FcγR^+/+ DC and 10 × 10⁶ CFSE – labeled 2D2 CD4 T cells were given PBS (No antigen), 600 μg agg Ig-MOG (tolerized), or 600μg sol Ig-MOG plus 33 μg CPG-ODN the next day (immunized). Forty-eight hours later, lymph node cells were stained with antibodies to the activation markers CD25, CD62L and CD44. Activation marker expression was assessed on CFSE⁺ cells. In (C) both the DC and 2D2 cells were unlabeled and analysis of marker expression was made on Vα3.2⁺ T cells under both immunizing and tolerizing conditions. The data is representative of 3 independent experiments with 2 mice per condition.

Figure 6. agg Ig-MOG treatment induces Th2 cytokines while sol Ig-MOG induces Th1

FcγR^−/− mice recipient of 6 × 10⁶ unlabeled FcγR^+/+ DC alone or in combination with 10 × 10⁶ unlabeled 2D2 CD4 T cells were on the next day given saline, 600 μg agg Ig-MOG (Agg), or 600 μg sol Ig-MOG (Sol) plus 33 μg CPG-ODN (CpG). Forty-eight hours later, lymph node cells were stimulated in vitro with 30 μg/ml MOG peptide (Filled bars) or 15 μg/ml PLP1 peptide as control (open bars) in the absence or presence of 20 μg/ml anti-IL-4R antibody (αIL-4R) (left column). In this case anti-IL-4R is used to prevent reabsorption by T cells. Cytokine production was measured by ELISA (left column) and ELISPOT (right column) for IFNγ (A), IL-5 (B), and IL-4 (C) as described in materials and methods. Each bar represents the mean ± SD of triplicate wells from 3 to 4 mice. N.D, not done.

Figure 7. Differential motility kinetics between immune Th1 and tolerant Th2 cells

FcγR^−/− mice were adoptively transferred with 10 × 10⁶ CFSE-labeled 2D2 CD4+ T cells and 6 × 10⁶ FcγR^+/+ DC. The next day the hosts were given i.p. 600 μg agg Ig-MOG tolerogen in saline (TOL), 600 μg sol Ig-MOG plus 33μg CPG-ODN in saline (IMM), or saline without Ig-MOG (NIL). The axillary and inguinal LNs were harvested at 36hr post antigen injection and subjected to 2-photon imaging. The behaviors of the 2D2 T cells were analyzed in detail using the Imaris software program (Bitplane). The graphs illustrate the (A) velocity, (B) motility coefficient, and (C) meandering index of live cells with movement tracks of 8 frames or higher. The scatter plots show representative lymph nodes for each condition. The bar graphs show the cumulative events collected from the lymph nodes of 3 mice per condition. The line represents the mean of analyzed cells. *p<0.05 as analyzed by one-way ANOVA.