Myelin antigen load influences antigen presentation and severity of central nervous system autoimmunity

Abstract

This study was designed to understand the impact of self-antigen load on manifestation of organ specific autoimmunity. Using a transgenic mouse model characterized by CNS hypermyelination, we show that larger myelin content results in greater severity of experimental autoimmune encephalomyelitis attributable to an increased number of microglia within the hypermyelinated brain. We conclude that a larger self-antigen load affects an increase in number of tissue resident antigen presenting cells (APCs) most likely due to compensatory antigen clearance mechanisms thereby enhancing the probability of productive T cell-APC interactions in an antigen abundant environment and results in enhanced severity of autoimmune disease.

Figure 1. Plp-Akt-DD transgenic mice develop more severe EAE compared to WT littermates

WT and Plp-Akt-DD transgenic female mice (n=8) were induced with EAE. Mice were weighed and evaluated daily for neurologic signs over a period of 30 days after immunization. Compared to WT mice transgenic mice showed significantly more severe disease as determined by (a) significantly greater increase in mean clinical scores over time (p<0.026; z score 2.218) (b) reduction in body weight (gms) during the course of disease progression (p<1.6×10⁻⁴). Error bars depict ±SE

Figure 2. Significantly increase CD3 positive T cell infiltrates are seen in the brain of Plp-Akt-DD transgenic mice compared to WT during acute and chronic phases of EAE

At peak of EAE onset (a) and during chronic EAE, 8 weeks post onset of EAE (b and c), brain tissue from Plp-Akt-DD transgenic and WT mice was perfused and sliced into 10μm frozen sections. Immunohistochemical staining with CD3 antibody shows significantly increased CD3+ mononuclear cell infiltrates around the perivascular spaces in the brain of Plp-Akt-DD transgenic mice (left panel) compared to WT (right panel) during acute phase of EAE (a) and progressing during the chronic phase of the disease to the hippocampus region extending into the brain parenchyma (b) as well as the fimbria and adjoining parenchyma (c). Representative sections presented in figure 2b and 2c to depict brain infiltration during chronic EAE were derived from mice with clinical scores ranging from 3-3.5 for Plp-Akt-DD transgenics and 0-3.5 for WT mice. Size bar =100 μm.

Figure 3. Alterations in brain morphology can be documented by MRI in Plp-Akt-DD transgenic mice with EAE

Eight weeks post onset of EAE (chronic phase EAE) Plp-Akt-DD transgenic mice with and without EAE, were imaged using a 7T magnet. T2 weighted MRI images (n=2) show visibly larger ventricle diffusion areas in Plp-Akt-DD transgenic mice (upper panel) compared to WT littermates with similar disease severity (lower panel). The relative brain to ventricular volume ratio in Plp-Akt-DD transgenic animals, quantified by drawing ROIs for the outer ventricle diffusion areas and the brain was found to be 0.0202 compared to 0.0071 in WT littermates reflecting an increase of 185% in transgenic animals consistent with substantial loss of brain parenchyma.

Figure 4. Plp-Akt-DD transgenic and wild type littermates have similar peripheral lymph node recall responses to the priming PLP 104-117 antigen

Ten days after priming with PLP 104-117 peptide, lymph node cells from Plp-Akt-DD transgenic and WT female mice (n=5) were restimulated with PLP 104-117 or OVA peptide in precoated ELISPOT plates for 72 hours. No significant differences were found in peripheral recall immune responses to the priming PLP 104-117 epitope between Plp-Akt-DD transgenic and WT animals as evidenced by frequency of (a) IL-17 (p=0.98) and (b) IFNγ (p=0.947) producing lymph node cells. Error bars depict +/− standard error.

Figure 5. Mononuclear cell population derived from the naïve Plp-Akt-DD transgenic brain can activate T cells with higher efficiency compared to that derived from the WT brain

Naïve Plp-Akt-DD and WT female mice were perfused through the intracardiac route with PBS (n=8). Mononuclear cells were extracted from brain homogenates by discontinuous density gradient centrifugation on a 30%-70% percoll gradient of brain homogenates and further activated with 10 ng/ml of IFNγ overnight. Pooled mononuclear cell preparation from n=8 naïve mice was used as antigen presenting cells (APC) to primed T cells derived from wild type littermates. T cells were purified using Thy1.2 positive magnetic beads from PLP 104-117 primed, ten day peripheral lymph nodes from WT SWR female mice. Brain derived mononuclear cells from naïve Plp-Akt-DD transgenic and wild type littermates and 3×10⁵ primed T cells from wild type mice were co-cultured at ratios 1:5, 1:10, 1:20 and 1:40 (APC:T cells) in antibody coated ELISPOT plates. Equal numbers of naïve Plp-Akt-DD transgenic brain derived mononuclear cells used as APCs can elicit significantly stronger recall responses from primed T cells when compared to mononuclear cells derived from naïve wild type animals as evident by higher frequencies of (a) IL-17 (p=0.036) and (b) IFNγ (p=0.038) spot forming units per 2×10⁵ T cells after 72 hours of culture.

Figure 6. Microglia selected from naïve Plp-Akt-DD transgenic or WT brain do not differ in their antigen presenting capacity and elicit similar T cell recall responses

Microglia were purified using CD11b coated magnetic beads from the mononuclear cell population derived from brains of naïve Plp-Akt-DD transgenic and WT animals (n=8). Purified and pooled populations of positively selected microglia were cultured overnight with (a) or without (b) 10 ng/ml IFNγ. Defined ratios of naïve microglia to PLP-104-117 primed peripheral T cells from WT mice were co-cultured in antibody pre-coated ELISPOT plates and evaluated for presence of IL-17 specific spot forming units. No significant differences were observed in primed T cell recall responses when antigen was presented by equal numbers of (a) activated or (b) non activated-Plp-Akt-DD transgenic or WT derived microglia (p=0.66 and 0.8 respectively).

Figure 7. Higher numbers of activated infiltrating T cells are observed in the brain of Plp-Akt-DD transgenic mice during EAE compared to wild type animals with EAE

At peak of EAE onset, brain infiltrating T cells were isolated from WT and Plp-Akt-DD mice (n=6) using percoll density gradient centrifugation and further enrichment by nylon wool columns. Infiltrating T cells from different mice were pooled together and cultured in IFNγ antibody coated ELISPOT plates for 72 hours without any antigenic stimulation and frequencies of IFNγ spot forming units were determined. Higher frequencies of IFNγ producing T cells were observed in the brain infiltrates derived from Plp-Akt-DD transgenics compared to those derived from WT littermates.

Figure 8. Plp-Akt-DD transgenic mice have increased numbers of CD45, CD11b and MHC class II positive cells compared to SWR WT mice

Mononuclear cells were isolated from the brain of naïve Plp-Akt-DD transgenic and naïve WT female mice and stained for CD11b-FITC and CD45-FITC microglial surface markers. (a) Flow cytometry analysis shows an approximate two fold increase in number of CD11b (left panel) and CD45 (right panel) positive cells in the naïve PLPAkt-DD transgenic brain (black line) compared to naïve WT animals (grey line). An increase in number of microglia was demonstrated in the naïve PLP-Akt-DD transgenic brain compared to naïve wild type animals by (b) immunohistochemical staining for Iba1expression in the hippocampal region (c) Real time quantitative PCR for Iba1 gene expression normalized to GAPDH expression (n=4; p<0.029). The expression of Iba-1 mRNA in the WT brain has been assigned an arbitrary value of 1.