B cells are capable of independently eliciting rapid reactivation of encephalitogenic CD4 T cells in a murine model of multiple sclerosis

<div><p>Recent success with B cell depletion therapies has revitalized efforts to understand the pathogenic role of B cells in Multiple Sclerosis (MS). Using the adoptive transfer system of experimental autoimmune encephalomyelitis (EAE), a murine model of MS, we have previously shown that mice in which B cells are the only MHCII-expressing antigen presenting cell (APC) are susceptible to EAE. However, a reproducible delay in the day of onset of disease driven by exclusive B cell antigen presentation suggests that B cells require optimal conditions to function as APCs in EAE. In this study, we utilize an <i>in vivo</i> genetic system to conditionally and temporally regulate expression of MHCII to test the hypothesis that B cell APCs mediate attenuated and delayed neuroinflammatory T cell responses during EAE. Remarkably, induction of MHCII on B cells following the transfer of encephalitogenic CD4 T cells induced a rapid and robust form of EAE, while no change in the time to disease onset occurred for recipient mice in which MHCII is induced on a normal complement of APC subsets. Changes in CD4 T cell activation over time did not account for more rapid onset of EAE symptoms in this new B cell-mediated EAE model. Our system represents a novel model to study how the timing of pathogenic cognate interactions between lymphocytes facilitates the development of autoimmune attacks within the CNS.</p></div

B cells are not sufficient APCs to support spontaneous EAE or optic neuritis.

<p>B cells are not sufficient APCs to support spontaneous EAE or optic neuritis.</p

B cells are capable of independently eliciting rapid reactivation of encephalitogenic CD4 T cells in a murine model of multiple sclerosis - Fig 5

<p><b>Flow cytometric analysis of lymphocytes from brains (left) and spinal cords (right).</b> (A) Mean ± SEM frequency of B cells and donor CD4 T cells as a percent of total mononuclear cells in the CNS of mice at week 1 (circles), week 2 (squares), or week 3 (triangles) post CD4 T cell transfer. Data is pooled from CD20-B^MHCIIxIgH^MOG mice, UBC^MHCII mice, and IAß^bstop^flox/floxxIgH^MOG (Cre^-) littermate controls from 9 different experiments with n = 3–5 mice at each time point prior to Tam treatment. Significance determined by Kruskal-Wallis test and Dunn’s correction for multiple comparisons. (B-D) Mean ± SEM frequency of B cells and donor CD4 T cells as a percent of total mononuclear cells in the brains (left) and spinal cords (right) harvested UBC^MHCII (red squares) and CD20-B^MHCIIxIgH^MOG (blue circles) mice approximately three days post EAE onset when mice were treated with Tam at (B) week 1, (C) week 2, or (D) week 3 post encephalitogenic CD4 T cell transfer. Data is pooled from 9 different experiments with n = 1–5 mice per genotype at each time point evaluated. Significance determined by Mann-Whitney test with two-tailed p value.</p

Accelerated EAE is not induced by CD4 T cells harvested from MHCII-deficient hosts.

<p>Accelerated EAE is not induced by CD4 T cells harvested from MHCII-deficient hosts.</p

Tam-inducible MHCII expression models recapitulate WT and B-cell mediated adoptive transfer EAE models.

<p>(A) Mean ± SEM EAE scores recorded for CD20-B^MHCIIxIgH^MOG (blue circles), and UBC^MHCII (red squares) mice treated with Tam by oral gavage and WT (black squares), CD19-B^MHCIIxIgH^MOG (grey circles), treated with corn oil vehicle by oral gavage once, three days prior to receiving 5x10⁶ encephalitogenic CD4 T cells. Data is representative of three independent experiments with n = 3–5 mice per genotype. (B) Day of EAE onset post cell transfer for WT (black squares), UBC^MHCII (red squares), CD19-B^MHCIIxIgH^MOG (grey circles) treated with corn oil 72 hours prior to CD4 T cell transfer and CD20-B^MHCIIxIgH^MOG (blue circles) recipient mice treated with Tam 72 hours prior to CD4 T cell transfer. Graph shows mean day of onset ± SEM from two pooled, independent experiments with n = 2–4 mice per genotype. Unpaired t test performed to compare day of onset between WT and CD19-B^MHCIIxIgH^MOG mice. (C) Mice were treated with Tam 72 hours prior to CD4 T cell transfer. Time to EAE onset for WT (black) and UBC^MHCII (red) mice is not significantly different by log-rank test (p = 0.107). Time to EAE onset for CD19-B^MHCIIxIgH^MOG (grey), and CD20-B^MHCIIxIgH^MOG (blue) mice is not significantly different by log-rank test (p = 0.481). Incidence curves generated from two pooled, independent experiments with n = 2–4 mice per genotype.</p

Inflammation and demyelination is not evident in spinal cords of encephalitic CD4 T cell recipients prior to Tam administration.

<p>(A) Representative spinal cord sections from recipients of encephalitogenic CD4 T cells (n = 5, CD20-B^MHCIIxIgH^MOG mice; n = 6, UBC^MHCII mice) were stained with Luxol Fast Blue, scale bar = 100um; all images generated from 10x magnification. (A) Spinal cords from CD20-B^MHCIIxIgH^MOG mice harvested three weeks after T cell transfer and before Tam administration (left). CD20-B^MHCIIxIgH^MOG mice treated with Tam three weeks after T cell transfer (middle) and harvested three days post EAE onset. UBC^MHCII mice treated with Tam three weeks after T cell transfer (right) and harvested three days post EAE onset. (B) Representative spinal cord sections from recipients of encephalitogenic CD4 T cells (at least mice 5 per genotype) were stained with antibodies to detect MOG, scale bar = 100um. Spinal cords from CD20-B^MHCIIxIgH^MOG mice (top left) and UBC^MHCII mice (bottom left) harvested three weeks after T cell transfer and before Tam administration. CD20-B^MHCIIxIgH^MOG (top right) and UBC^MHCII (bottom right) mice treated with Tam three weeks after T cell transfer and harvested three days post EAE onset. (C) Regions from rostral to caudal sections of spinal cords from CD20-B^MHCIIxIgH^MOG (blue) and UBC^MHCII mice (red) were harvested and scored for inflammation. Graph shows mean +/- SEM inflammation scores and Kruskal-Wallis test with Dunn’s correction for multiple comparisons was applied. B.S. = brainstem; C = cervical; T = thoracic; L = lumbar. (D) Mean (SD) percent area of demyelinated white matter was quantified for thoracic spinal cord sections from CD20-B^MHCIIxIgH^MOG (blue) and UBC^MHCII (red) mice. Significance determined by Mann-Whitney test with two-tailed p value.</p

Tam treatment after T cell transfer results in accelerated B cell mediated EAE.

<p>(A) Mean ± SEM day of EAE onset for WT (black squares) and CD19-B^MHCIIxIgH^MOG (grey circles) post T cell transfer, and for UBC^MHCII (red squares) and CD20-B^MHCIIxIgH^MOG (blue circles) treated with Tam at either one week, two weeks, or three weeks post T cell transfer. Data is pooled from 12 different experiments with n = 1–5 mice per genotype and time point evaluated, p values calculated by unpaired t tests. (B) Time to EAE onset is not significantly different for UBC^MHCII mice (left graph) treated with Tam at either Week 1 (solid line), Week 2 (dashed line), or Week 3 (dotted line) post CD4 T cell transfer. Time to EAE onset is significantly delayed for CD20-B^MHCIIxIgH^MOG mice (right graph) treated with Tam at either Week 1 (solid line), compared to Week 2 (dashed line), or Week 3 (dotted line) post CD4 T cell transfer. Incidence curves generated from is pooled from 12 different experiments with n = 1–5 mice per genotype and time point, with significance evaluated by log-rank test.</p

B cells are capable of independently eliciting rapid reactivation of encephalitogenic CD4 T cells in a murine model of multiple sclerosis - Fig 6

<p>(A-D) Mean ± SEM of the geometric mean fluorescence intensity of activation marker expression on donor CD4 T cells harvested from spleens of recipient mice at week 1 (circles), week 2 (squares), or week 3 (triangles) post CD4 T cell transfer. Data is pooled from CD20-B^MHCIIxIgH^MOG mice, UBC^MHCII mice, and IAß^bstop^flox/floxxIgH^MOG (Cre^-) littermate control recipients from five independent experiments with n = 2–4 mice per time point evaluated. Significance tested by one way ANOVA with Tukey’s test for multiple comparisons. Geometric mean fluorescence intensity of (A) CD44, (B) CD62L, (C) CD25, and (D) CD69 expression on donor CD4 T cells. (E) Frequency of IFNγ-expressing donor CD4 T cells pre-transfer and following <i>in vivo</i> incubation for three weeks in MHCII-deficient hosts. Graph is representative of n = 5 independent experiments with at least two mice from each genotype. Donor T cells were harvested from the spleen. Significance tested by Kruskal-Wallis test and Dunn’s correction for multiple comparisons.</p

Temporal regulation of MHCII expression <i>in vivo</i>.

<p>(A) MHCII expression on peripheral blood B cells collected from UBC^MHCII (red line) and CD20-B^MHCIIxIgH^MOG (blue shaded) at several time points after Tam administration. Data are representative of two experiments, (n = 2 mice per time point from each genotype). (B) 72 hours post Tam treatment, spleens were harvested from naïve mice 72 hours after oral gavage with Tam for UBC^MHCII (red squares) and CD20-B^MHCIIxIgH^MOG (blue circles) or corn oil vehicle for WT (black squares) and CD19-B^MHCIIxIgH^MOG mice (grey circles). The frequency of MHCII+ B cells as a percentage of total B cells is presented as mean ± SEM from n = 3 mice per genotype.</p