CD5-CK2 binding/activation-deficient mice are resistant to experimental autoimmune encephalomyelitis: protection is associated with diminished populations of IL-17-expressing T cells in the central nervous system

Abstract

Regulating the differentiation and persistence of encephalitogenic T cells is critical for the development of experimental autoimmune encephalomyelitis (EAE). We reported recently that CD5 has an engagement-dependent prosurvival activity in T cells that played a direct role in the induction and progression EAE. We predicted that CD5 regulates T cell apoptosis/survival through the activation of CK2, a prosurvival serine/threonine kinase that associates with the receptor. To test this hypothesis, we generated mice expressing CD5 with the inability to bind and activate CK2 and assessed their susceptibility to EAE. We found mice deficient in CD5-CK2 signaling pathway were mostly resistant to the development of EAE. Resistance to EAE was associated with a dramatic decrease in a population of effector infiltrating Th cells that coexpress IFN-gamma and IL-17 and, to a lesser extent, cells that express IFN-gamma or IL-17 in draining lymph nodes and spinal cords. We further show that T cells deficient in CD5-CK2 signaling hyperproliferate following primary stimulation; however, following restimulation, they rapidly develop nonresponsiveness and exhibit elevated activation-induced cell death. Our results provide a direct role for CD5-CK2 pathway in T cell activation and persistence of effector T cells in neuroinflammatory disease. This study predicts that targeting of IFN-gamma(+)/IL-17(+) infiltrating Th cells will be useful for the treatment of multiple sclerosis and other systemic autoimmune diseases.

FIGURE 1

Generation and phenotypic characterization of CD5Δ458-461 mice. A, CD5WT and CD5Δ458-461 CD2 minigene constructs. *The CD5WT transgenic mouse as well as the CD2 minigene construct was generously provided to us by Dr. Paul Love, NIH (10). B-C, Frequencies of CD4⁺ and CD8⁺ lymphocytes in thymus and spleen of 6-8 week old mice. Percentage of cells in each quadrant is given. D-E, Expression levels of CD5 on T-cell subsets in the thymus and spleen.

FIGURE 2

CD5-CK2 binding/activation deficient mice are resistant to EAE. A, Clinical course of MOG_35-55-induced EAE in CD5^+/+ (N=18), CD5^-/- (N=16), CD5WT (N=9) and CD5Δ458-461 (N=13) mice. EAE was induced in mice following s.c. immunization with 150μg MOG_35-55 as described in Materials and Methods. Results are the mean clinical score (bars represent SEM). B, Mean cumulative index: the mean of the sum of the daily clinical scores observed between days 1 and 30. C, Mean maximum clinical score: calculated from the maximum clinical score of individual animals between day 1 and 30. D, Disease incidence: represents the percentage of mice developing EAE. Mice with a clinical score ≥2 are considered having clinical disease. * P<0.05 with Student’s t-test.

FIGURE 3

CD4 T-cell populations during acute EAE. DLN cells from CD5^+/+, CD5WT, CD5^-/- and CD5Δ458-461 mice were analyzed for changes in frequency of CD4⁺CD69⁺ T-cells (4) from 4 to 12 days following s.c. immunization with MOG_35-55 peptide; time points representing early activation of acute phase of the disease. B, CD4⁺ T-cell infiltration in spinal cords of CD5^+/+, CD5WT CD5^-/- and CD5Δ458-461 mice 9 to 17 following immunization with MOG peptide. C. CD8⁺ and CD11b⁺_35-55 populations in spinal cords 12 days following immunization. Mononuclear cells were prepared from perfused animals as described in Materials and Methods.

FIGURE 4

Histology of spinal cord from CD5WT and CD5Δ458-461 mice 17 days after immunization with MOG_35-55 peptide. Serial sections of spinal cord stained with hematoxylin and eosin (H&E) (A) and Luxol fast blue (B). H&E staining section shows infiltration of mononuclear cells in grey matter of both CD5WT and CD5Δ458-461 mice and white matter of CD5WT mice (arrow). Luxol fast blue staining shows areas of intact myelin (blue) and demyelination (pink). Arrows indicate demyelination in CD5WT cord which include sites associated with infiltration of mononuclear cells in myelin area.

Figure 5

Spinal cords of CD5Δ458-461 mice contain greater frequency of cycling (BrDU⁺) CD4 T-cells. To determine frequency of cycling CD4 T-cells, MOG-immunized CD5WT and CD5Δ458-461 mice were injected with 1.5 mg BrDU i.p 12 h before sacrifice on day 17 and BrDU incorporation was determined by flow cytometry. Data is a representation of 2-3 experiments.

FIGURE 6

Populations of effector TH cells in DLN and spinal cords of MOG_35-55 immunized mice. Intracellular staining of CD4⁺ T-cells for γIFN and IL-17 in DLN (day 4, 9) and spinal cord mononuclear cells (day 9 and 17). A lymphocyte gate was used for all analyses. The unbracketed numbers in each quadrant represent the frequency of CD4⁺ T-cells expressing γIFN and/or IL-17. The bracketed numbers (day 17 spinal cord) represent normalized values to take into consideration the differences in absolute numbers of CD4⁺ T-cells between CD5WT and CD5Δ458-461 mice. The normalized numbers represent the frequency of cytokine expressing CD4⁺ T-cells in the lymphocyte gate. Each data point represents a pool of 3-8 mice from 2-3 experiments.

FIGURE 7

CD5-CK2 binding/activation deficient T-cell hyperproliferate following in vitro stimulation. A, Frequency of CD4⁺ and CD8⁺ T-cells incorporating BrDU at 72 h of anti-CD3 stimulation (1μg/ml). Cells were pulse labeled with BrDU for the last 90 min before analysis. B, Number of cell divisions was measured by CFSE dilution assay after 72 h of anti-CD3 stimulation (1μg/ml). The frequency of cells in each CFSE-dilution peak is given.

Figure 8

CD5-CK2 binding/activation deficient T-cells have decreased viability after prolonged exposure to stimulation. A, [³H]thymidine incorporation was determined at 24, 48, 72 and 96 h of anti-CD3 stimulation (1μg/ml). B, Percentage of apoptotic (DiOC₆^lo) CD4⁺ and CD8⁺ cells after 24 of anti-CD3 stimulation. Data is a representation of 3 experiments.

FIGURE 9

CD5-CK2 binding/activation deficient T-cells die at a higher rate than CD5WT T-cells and develop non-responsiveness following restimulation in recall response assay. After initial stimulation with 1μg/ml of anti-CD3 for 24 h, lymphocytes were rested for 72 h before re-stimulation with anti-CD3 (1μg/ml). A, Frequency of CD4⁺ and CD8⁺ cells in cycle (BrDU⁺) (see Figure 7 legend and Materials and Methods) after 48 h of re-stimulation. B. Percent apoptotic (DiOC₆^lo) CD4⁺ and CD8⁺ cells after 48 h of restimulation. Data is a representation of 3 experiments.