Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation

Abstract

Cytomegalovirus (CMV) is a latent virus which causes chronic activation of the immune system. Here, we demonstrate that cytotoxic and pro-inflammatory CD4⁺CD28^null T cells are only present in CMV seropositive donors and that CMV-specific Immunoglobulin (Ig) G titers correlate with the percentage of these cells. In vitro stimulation of peripheral blood mononuclear cells with CMVpp65 peptide resulted in the expansion of pre-existing CD4⁺CD28^null T cells. In vivo, we observed de novo formation, as well as expansion of CD4⁺CD28^null T cells in two different chronic inflammation models, namely the murine CMV (MCMV) model and the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis (MS). In EAE, the percentage of peripheral CD4⁺CD28^null T cells correlated with disease severity. Pre-exposure to MCMV further aggravated EAE symptoms, which was paralleled by peripheral expansion of CD4⁺CD28^null T cells, increased splenocyte MOG reactivity and higher levels of spinal cord demyelination. Cytotoxic CD4⁺ T cells were identified in demyelinated spinal cord regions, suggesting that peripherally expanded CD4⁺CD28^null T cells migrate towards the central nervous system to inflict damage. Taken together, we demonstrate that CMV drives the expansion of CD4⁺CD28^null T cells, thereby boosting the activation of disease-specific CD4⁺ T cells and aggravating autoimmune mediated inflammation and demyelination.

Figure 1

CMV infection expands CD4⁺CD28^null T cells. Flow cytometry was performed to determine the percentage of CD4⁺CD28^null T cells. CMV and EBV status and immunoglobulin titers were determined via ELFA. CD4⁺CD28^null T cells in (a) CMV seropositive (n = 100) compared to seronegative (n = 127) MS patients and (b) CMV seropositive (n = 24) versus seronegative healthy controls (n = 39). (c) Correlation of CMV IgG levels with the percentage of CD4⁺CD28^null T cells in MS patients and HC (n = 140). (d) Correlation of EBV EBNA IgG titers in 155 MS patients. (e) Repeated stimulation of PBMCs from HC (n = 12) and MS patients (n = 8) with CMV pp65 (e) or IL-2 (f) in vitro, after which the number of CD4⁺CD28^null T cells was determined at different time points. (g) Flow cytometry of splenocytes of MCMV infected mice on day 0, day 8 and day 250 post infection (n = 5/time point). (h) Splenocytes from MCMV-infected WT (n = 5) and CD80/86^−/− (n = 4) mice were analysed for the percentage of CD4⁺CD28^null T cells at day 250 post infection. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2

CD4⁺CD28^null T cells are increased in EAE mice, as a result of auto-antigenic stimulation. (a) The phenotype of blood-derived mouse CD4⁺CD28^null T cells was measured via flow cytometry. (b) After induction, EAE mice (n = 15) and CFA control mice (n = 10) were scored for maximum 90 days according to their disability. (c) Blood was collected at different time points, to determine the number of CD4⁺CD28^null T cells via flow cytometry. (d) Correlation between CD4⁺CD28^null T cells and EAE score. (e) Historical human MBP specific T cell clones repeatedly stimulated with MBP/PHA (n = 8) were thawed and analysed for the number of CD4⁺CD28^null T cells via flow cytometry. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

MCMV infected mice with EAE have a worse disease course. Mice were infected with MCMV and after 8 days EAE was induced. (a) Daily scoring of the CMV, EAE and MCMV infected EAE groups (n = 8/group). (b) Splenocytes were isolated at day 0 (baseline) and day 30 and CD4⁺CD28^null T cells were measured via flow cytometry. (c) After stimulation of splenocytes with a MOG_35–55 peptide, the IFNγ producing CD4⁺ T cells were measured via flow cytometry and normalized to the non-peptide control. The MOG response significantly correlated with the percentage of CD4⁺CD28^null T cells in the spleen. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

CMV infection increases EAE lesion size. (a) Representative staining of MBP in the CMV control group (left), the EAE control group (middle) and the CMV infected EAE animals (right). (b) The amount of demyelination within the CMV and EAE control groups and the CMV infected EAE group, calculated via dividing the demyelinated area (=loss of MBP) in the white matter of the spinal cord, over the total white matter area for each section. (c) Correlation between the amount of demyelination for each animal and the percentage of peripheral CD4⁺CD28^null T cells. (d) Single and double staining of CD4 and Granzyme B, which points to the presence of CD4⁺CD28^null T cells, in the spinal cord of EAE mice. *p < 0.05, ****p < 0.0001.