Lymph node-derived donor encephalitogenic CD4+ T cells in C57BL/6 mice adoptive transfer experimental autoimmune encephalomyelitis highly express GM-CSF and T-bet

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 μg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.

Figure 1

General schematic for induction of EAE by adoptive transfer into C57BL/6 mice. Groups of donor mice were immunized with CFA/(MOG)_35-55. Some mice also received pertussis toxin (PT) on the day of immunization (day 0) and on day 2 post immunization. Lymph node cells (LNC) and splenocytes SPC) were harvested at either day 6 or day 12 post immunization and were restimulated in vitro with a combination of MOG_p35-55 and IL-2, or MOG_p35-55 and IL-12 for 3-8 days in tissue culture flasks. After specified days in culture, 10-20 × 10⁶ cells in 200 μL PBS were injected into recipient mice. Mice were observed every day after injection for disease onset and clinically scored.

Figure 2

EAE disease course in C57BL/6 mice after adoptive transfer of day 12 LNC. The successful protocol required LNC that were harvested from donor mice that had been immunized 12 days previously and had been treated with PT. Following LNC isolation, the cells were restimulated in vitro with MOG_p35-55 (10 μg/ml) and IL-12 (0.5 ng/ml) for 72 hours. Error bars represent ± SD. Clinical scores shown are from at least two independent experiments. The difference between the successful experimental paradigm (●)and all unsuccessful paradigms (O) with regard to disease activity was significant, starting on day 7 (p ≤ 0.001).

Figure 3

Histopathological examination of the CNS of donor mice. Representative data on the inflammatory infiltrates in the brain and spinal cord on day 12 are shown. A. Cerebellar cortex. B. Lateral brainstem. C. Posterior colliculus. D. Ventral brainstem. E. H&E and F. LFB/PAS/H&E of spinal cord dorsal root. G. H&E and H. LFB/PAS/H&E of lateral spinal cord white matter. I. H&E and J. LFB/PAS/H&E of ventral spinal cord white matter.

Figure 4

Determining cytokine secretion by in vivo polarized T lymphocytes. LNC and SPC from donor mice immunized 6 or 12 days previously were stimulated directly ex-vivo with PMA (50 ng/ml) and ionomycin (750 ng/ml) to determine the frequency of recently activated CD3⁺CD4⁺CD44⁺ T cells present in these tissues prior to in vitro restimulation with antigen. IFNγ and Il-17 expressing T cells in LNC and SPC at day 6 and day 12 are shown in panels B, D, F, and H. Isotype controls are shown in panels A, C, E, and G. Data are representative of at least three independent experiments.

Figure 5

Cytokine production in vitro. LNC and SPC harvested from donor mice at day 6 and day 12 post-immunization were cultured in vitro with 10 μg/ml MOG_35-55 with 0.5 ng/ml IL-12. The concentration of IFN-γ (A), IL-17 (B), and GM-CSF (C) secreted by spleen and LNC after 72 hours in culture was determined by cytokine bead array (CBA). Data shown are representative of at least 2 independent experiments. ns = not significant.

Figure 6

Flow cytometry analysis of expression of T-bet by encephalitogenic CD4⁺ T cells is maintained in Day 12 LNC. C57BL/6 donor mice were immunized with CFA/MOG_p35-55 followed by PT at day 0 and day2. At day 6 or day 12, LNC and SPC were harvested and cultured for 72 hours in the presence of MOG_p35-55 and IL-12 and prepared for flow cytometry. CD3⁺CD4⁺CD44⁺ T cells were examined for expression of T-bet. The percentage of T-bet⁺ cell is indicated in the quadrants B, D, F, & H. Isotype controls are shown in panels A, C, E, and G. Data shown are representative of at least 3 independent experiments.

Figure 7

Determining MOG-specificity in donor LNC and SPC. At day 6 or day 12, LNC and SPC were harvested and cultured for 72 hours in the presence of MOG_p35-55 and IL-12 and prepared for tetramer staining. CD3⁺CD4⁺CD44⁺ T cells were examined for staining with MOG_38-49 MHC class II tetramer, or control MHC class II tetramers containing hCLIP_103-117. The percentage of MOG_38-49 MHC class II tetramer positive cell is indicated in the regions B, D, F, & H. Control MHC class II tetramers containing hCLIP_103-117 staining is shown in panels A, C, E, and G. Data shown are representative of at least 3 independent experiments.