Cutting Edge: Conditional MHC Class II Expression Reveals a Limited Role for B Cell Antigen Presentation in Primary and Secondary CD4 T Cell Responses

Angela S. Archambault, Javier A. Carrero, Lisa G. Barnett, Nigel G. McGee, Julia Sim, Jonathan O. Wright, Tobias Raabe, Peiquin Chen, Hua Ding, Eric J. Allenspach, Ioannis Dragatsis, Terri M. Laufer and Gregory F. Wu
J Immunol July 15, 2013, 191 (2) 545-550; DOI: https://doi.org/10.4049/jimmunol.1201598

Abstract

The activation, differentiation, and subsequent effector functions of CD4 T cells depend on interactions with a multitude of MHC class II (MHCII)–expressing APCs. To evaluate the individual contribution of various APCs to CD4 T cell function, we have designed a new murine tool for selective in vivo expression of MHCII in subsets of APCs. Conditional expression of MHCII in B cells was achieved using a cre-loxP approach. After i.v. or s.c. priming, partial proliferation and activation of CD4 T cells was observed in mice expressing MHCII only by B cells. Restricting MHCII expression to B cells constrained secondary CD4 T cell responses in vivo, as demonstrated in a CD4 T cell–dependent model of autoimmunity, experimental autoimmune encephalomyelitis. These results highlight the limitations of B cell Ag presentation during initiation and propagation of CD4 T cell function in vivo using a novel system to study individual APCs by the conditional expression of MHCII.

Introduction

Multiple APCs expressing MCH class II (MHCII) can engage in cognate interactions that are critical to the development, differentiation, and effector functions of CD4 T cells. Although dendritic cells (DCs) are recognized as potent initiators of CD4 T cell responses (1), Ag-specific B cells are actually more adept at acquiring and presenting soluble cognate Ags in vivo compared with DCs (2). Contributing to the complexity involved in MHCII-dependent responses in vivo is the substantial reliance to date on indirect experimental models that have limited the ability to discern the degree to which individual APC subsets orchestrate CD4 T cell function.

Traditionally, B cells have been considered accessory APCs to DCs (3). However, accumulating evidence suggests that B cells regulate Ag-specific CD4 T cell immune responses, such as priming and memory responses (4, 5). Potent regulatory and tolerogenic properties have also been attributed to B cells (6, 7). Furthermore, a role for B cell Ag presentation has been implicated during disease, because anti-CD20–mediated B cell depletion is an effective therapy for human autoimmune diseases such as multiple sclerosis, apparently independent of effects on Ab levels (8). Whether B cells alone are capable of directing cognate CD4 T cell behavior during autoimmunity has not been directly tested.

To examine the individual contribution of various APC subsets to CD4 T cell function, we have established a new in vivo system to conditionally express MHCII. In this article, we demonstrate that MHCII expression can be restricted to cell lineages using a cre-loxP system. Examining B cell Ag presentation, we found in vivo priming of CD4 T cells by B cells alone does occur but is limited. Moreover, secondary responses coordinated by B cells were also restricted, and B cell Ag presentation was not sufficient to support CD4 T cell–dependent autoimmune encephalomyelitis. These results demonstrate the limited sufficiency of B cell Ag presentation to direct CD4 T cell responses whereas providing evidence of the utility of this system for the study of individual APC contributions in vivo.

Materials and Methods

Mice

C57BL/6, B6.PL, 2D2, CMVCre, and CD19Cre mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MHCII−/− mice were used as described previously (9). A polyadenylation stop sequence flanked by loxP sites (10) was targeted to the first intron of the IAβ locus, making use of a retrieval vector PL253 and bacterial artificial chromosome recombineering (11). No endogenous sequences were deleted by the insertion. Mice bred for homozygosity of this construct are termed IAβbstopf/f. The final targeting vector was verified by sequencing of all essential elements, linearized, and electroporated into the LK1 C57BL6 ES cell line (12). Southern blot analysis confirmed appropriate targeting (Supplemental Fig. 1).

Abs and flow cytometry

Abs were purchased from BD Biosciences (San Jose, CA) and eBioscience (San Diego, CA). Samples were acquired on FACSCalibur or LSRII flow cytometers (BD, San Jose, CA) or Beckman Coulter Gallios (Brea, CA). Gating for percentage CFSE dilution was relative to the undivided peak in each individual experiment.

Cell purification, lines, and culture

CD4 T and B cells were positively selected using mouse L3T4 and CD19 microbeads (Miltenyi Biotec, Auburn, CA). RNA purification and RT-PCR were performed as described previously (13). A total of 1–5 × 106 B16 cells (14) were injected s.c. into mice to purify bone marrow (BM)–derived DCs (BMDCs) that were cultured in RPMI medium with 10% FBS and 10 ng/ml GM-CSF (R&D Systems). Peritoneal macrophages were isolated by peritoneal lavage from mice 3–5 d after i.p. injection of 40 μg Con A (Sigma, St. Louis, MO). T cell hybridomas were generated as previously described (15).

Proteins and peptides

MOG protein expression and purification were performed as reported previously (9). MOG35–55 was commercially synthesized (CSBio, Menlo Park, CA). Listeriolysin O peptide fragment 190–201 was synthesized at Washington University in St. Louis (16).

In vitro and in vivo proliferation experiments

Spleens were processed for B cell depletion by AutoMACS. Single-cell suspensions were irradiated with 2000 rad, washed, and then combined with Ag. A total of 1 × 105 APCs were cultured with 5 × 104 hybridomas and Ag overnight at 37°C. Proliferation of CTLL-2 was measured after addition of supernatant by [3H]thymidine incorporation (16). CD4 T cells were isolated from 2D2 mice and labeled with CFSE (Invitrogen, Grand Island, NY). For priming studies, 2 × 106 CFSE-labeled congenic (CD45.1) CD4 T cells were transferred i.v. 1 day before immunization.

ELISPOT assays

IFN-γ and IL-2 ELISPOT assays were performed as described previously (16) using 5 × 105 splenocytes/well in triplicate with 1 × 105 2D2 CD4 T cells and stimulated with no Ag or varying doses of MOG35–55/ml.

Immunizations and induction of experimental autoimmune encephalomyelitis

Thymic grafting was performed before the induction of active experimental autoimmune encephalomyelitis (EAE) as reported previously (9). Immunization i.v. with 50 μg MOG35–55 or 100 μg rMOG was done with 50 μg CpG (IDT, Coralville, IA). Active s.c. immunization with rMOG or MOG35–55 was performed as reported previously (9). Passive EAE was induced as described with 1 × 107 MOG-specific, Thy1.1+ encephalitogenic cells that are almost exclusively Th1 (13).

Statistics

Statistical analysis was performed using two-tailed Student t tests.

Results and Discussion

Conditional inactivation of IAβb in vivo results in abrogation of MHCII expression

To test the sufficiency of Ag presentation by specific lineages of APCs, we designed a mouse system in which MHCII is conditionally expressed. We successfully targeted a stop sequence flanked by loxP sites (10) to the IAβ locus (Supplemental Fig. 1). Southern blot analysis confirmed germline transmission of the construct in several founder mice. We examined mice homozygous for the insert, termed IAβbstopf/f mice, for expression of MHCII. In peripheral blood, spleen, and BM compartments, MHCII expression was abolished (Supplemental Fig. 1D). Consistent with the lack of MHCII and positive selection in the thymus, CD4 T cells were absent. By flow cytometry, splenic and BMDCs from mice treated with Flt3-ligand expressed no discernible MHCII (Supplemental Fig. 1E). Thus, we have successfully generated mice in which a removable knock-in stop construct in the IAβ chain locus eliminates MHCII expression as detected by FACS.

Conditional in vivo gene repair of IAβ in B cells

Our targeted insert results in elimination of MHCII expression but retains the capacity to re-express MHCII in a cell lineage–specific manner. To explore the contribution of B cell Ag presentation to CD4 T cell responses, we reconstituted MHCII expression using the CD19Cre mouse (17). CD19Cre and IAβbstopf/f mice bred to homozygosity for the IAβ allele, termed CD19Cre/IAβbstopf/f, were examined for MHCII expression in lymphoid organs. Expression of MHCII in B cells was identical in both CD19Cre/IAβbstopf/f and wild-type (WT) BM and spleen by FACS (Fig. 1A, Supplemental Fig. 2A). The development and functionality of B cells was not altered (Supplemental Fig. 2). In contrast, CD11c+CD19− cells were devoid of MHCII expression in CD19Cre/IAβbstopf/f mice. Of note, small fractions of CD19+ cells also express CD11c, CD11b, or both (18), and CD19+CD11c+ cells from CD19Cre/IAβbstopf/f mice were observed to be MHCII+. However, all CD11c+ cells from CD19Cre/IAβbstopf/f mice expressing MHCII were CD19+ (Supplemental Fig. 2E). We also examined the expression of MHCII in other subsets of APCs. WT BMDCs and peritoneal macrophages exhibited clear expression of MHCII. However, both types of APCs from IAβbstopf/f mice had no MHCII expression detectable by FACS, and only APCs expressing CD19 were MHCII+ in CD19Cre/IAβbstopf/f mice (Supplemental Fig. 2F).

FIGURE 1.
FIGURE 1.

MHCII expression is functionally abrogated in IAβbstopf/f mice and restricted to B cells in CD19Cre/IAβbstopf/f mice. MHCII expression was analyzed after crossing CD19Cre mice (17) to IAβbstopf/f mice. (A) BM (top) and spleen (bottom) expression of MHCII in IAβbstopf/f (shaded), CD19Cre/ IAβbstopf/f (blue line), and WT (black line) mice. Histograms are from singlet cells gated on CD11c+CD19− cells (left), and CD19int (middle) and CD19hi populations. Data are representative of six or more mice per group analyzed in three experiments. (B) BMDCs generated from IAβbstopf/f (gray), CD19Cre/IAβbstopf/f (white), and WT (black) mice were pulsed with MOG35–55 peptide or rMOG protein, then cocultured with the MOG-specific T cell hybridoma, MOG.15. IL-2 production was assayed by CTLL-2 proliferation assay using [3H]thymidine incorporation (16). (C) MOG35–55 was added to spleens from WT (black), IAβbstopf/f (red), and CD19Cre/IAβbstopf/f (blue) mice before (square) and after (circle) depletion of B cells. Further enrichment for DCs was also performed (X marker). CTLL-2 were incubated with supernatants from MOG.15 and splenic APC cultures with varying doses of MOG35–55. Data are representative of triplicate samples in two separate experiments.

To verify the functional degree to which MHCII expression is restricted to B cells in CD19Cre/IAβbstopf/f mice, we tested the ability of several APC subsets to present Ag to CD4 T cells. BMDCs from WT mice elicited a robust response to the immunodominant CD4 T cell peptide of MOG (MOG35–55) by an IAb-restricted CD4 T cell hybridoma, MOG.15. In contrast, BMDCs from either IAβbstopf/f or CD19Cre/IAβbstopf/f mice failed to generate Ag-specific responses (Fig. 1B). Thus, DCs from IAβbstopf/f and CD19Cre/IAβbstopf/f mice cannot generate MHCII-dependent CD4 T cell proliferation. To determine the level of functional splenic MHCII expression, we incubated irradiated splenocytes from WT, IAβbstopf/f, and CD19Cre/IAβbstopf/f mice with MOG35–55. The MOG-specific hybridoma, MOG.15, responded in a dose-dependent fashion to Ag with WT or CD19Cre/IAβbstopf/f splenocytes, but not to Ag with splenocytes from IAβbstopf/f mice (Fig. 1C). Importantly, removal of CD19+ cells from the spleen before incubation with MOG35–55 abrogated Ag presentation by CD19Cre/IAβbstopf/f, but not WT, splenocytes (Fig. 1C). These results confirm the absence of functionally relevant levels of MHCII expression in IAβbstopf/f mice and demonstrate the expression of MHCII in CD19Cre/IAβbstopf/f mice is restricted to B cells. Additional enrichment for CD11c+ cells did not result in any detectable Ag-specific response by either IAβbstopf/f or CD19Cre/IAβbstopf/f splenocytes (Fig. 1C). Similar results were obtained using the non-self Ag, listeriolysin O peptide fragment 190–201 (16) (Supplemental Fig. 2G).

B cells are capable of limited CD4 T cell priming

CD19Cre/IAβbstopf/f mice provide the optimal system in which to examine the in vivo capacity for B cells alone to drive peripheral CD4 T cell responses de novo. We assessed the ability of B cells to stimulate proliferation of MOG-specific 2D2 TCR transgenic CD4 T cells. MOG35–55 together with CpG was delivered i.v. to mice that had received 2D2 CD4 T cells labeled with CFSE. Virtually all 2D2 cells isolated from the spleen of WT mice exhibited some degree of proliferation after i.v. immunization (92.5 ± 10.9%). In contrast, no CD4 T cell proliferation was observed in the absence of MHCII (4.5 ± 2.7% in IAβbstopf/f mice, p < 0.01 versus WT), as expected because of dependence by rapid homeostatic proliferation on TCR–MHCII interactions. 2D2 CD4 T cells in CD19Cre/IAβbstopf/f mice proliferated after i.v. immunization with MOG35–55, but only 55.3 ± 7.1% diluted CFSE (p < 0.01 versus WT and versus IAβbstopf/f; Fig. 2A, 2C). Similar results were observed after protein administration (Fig. 2B, 2D). Examination of activation and differentiation markers revealed partial downregulation of CD62L on 2D2 cells in CD19Cre/IAβbstopf/f, but not IAβbstopf/f, mice, compared with WT mice after peptide, but not protein, immunization (Fig. 2G). No difference in other markers, including CD69, CD25, Foxp3, PD-1, or BTLA, was observed (data not shown).

FIGURE 2.
FIGURE 2.

B cells have a limited capacity to prime CD4 T cells in vivo. (A–F) CFSE-labeled 2D2 T cells were adoptively transferred into IAβbstopf/f (“F”; red), CD19Cre/IAβbstopf/f (“C”; blue), and WT (black) mice 1 d before immunization with 50 μg MOG35–55 i.v. (A) or 100 μg rMOG i.v. (B) and 50 μg CpG. Shown are representative FACS plots from at least two different experiments with three mice per group 3 d after immunization. Percent of dividing 2D2 cells is shown for i.v. peptide (C) or protein (D), or s.c. MOG35–55 (E) or rMOG s.c. (F) in CFA. (G) Percent donor 2D2 cells isolated from mice immunized with s.c. or i.v. MOG35–55, or s.c. or i.v. rMOG, with CD62Llow expression. Each graph represents at least three mice per group. *p < 0.001 (C–F) or p < 0.01 (G) in comparison with either other group; **p < 0.01 in comparison with WT (C, D) or IAβbstopf/f (G).

After s.c. immunization with MOG35–55, 83.3 ± 3.0% of 2D2 cells in the draining lymph node of WT mice exhibited CFSE dilution. In contrast, >90% of 2D2 cells remained undiluted in both IAβbstopf/f and CD19Cre/IAβbstopf/f mice (Fig. 2E). Minimal proliferation of 2D2 cells was induced in IAβbstopf/f or CD19Cre/IAβbstopf/f mice after protein immunization as well (Fig. 2F), demonstrating that MHCII expression by B cells does not contribute to Ag-specific CD4 T cells proliferation after s.c. immunization. However, 2D2 cells still exhibited a significant reduction in CD62L expression in CD19Cre/IAβbstopf/f compared with IAβbstopf/f mice after s.c. peptide immunization (20.2 ± 2.3 versus 7.6 ± 0.3%, respectively, p < 0.05; Fig. 2G).

Secondary CD4 T cell responses, including those critical for inducing autoimmune encephalomyelitis, are limited in CD19Cre/IAβbstopf/f mice

B cells participate in cognate interactions with CD4 T cells that appear essential to immune activity, autoimmunity, and tolerance (19, 20). To evaluate the ability of B cells alone to promote CD4 T cell function after the priming phase, we cocultured previously primed CD4 T cells with Ag and splenocytes from IAβbstopf/f, CD19Cre/IAβbstopf/f, or WT mice. ELISPOT analysis revealed an increase in Ag-specific IFN-γ production by 2D2 CD4 T cells cultured with splenocytes from WT mice compared with IAβbstopf/f splenocytes (p < 0.01; Fig. 3A). Cognate interactions between previously primed 2D2 CD4 T cells and splenocytes from CD19Cre/IAβbstopf/f mice also produced greater IFN-γ as compared with IAβbstopf/f (p < 0.01), but was reduced in comparison with WT (p < 0.05; Fig. 3A). In contrast, 2D2 production of IL-2 and IL-17 that was observed to result from cognate interactions with WT splenocytes was not elicited by splenocytes from IAβbstopf/f or CD19Cre/IAβbstopf/f mice. Conversely, Ag-specific production of GM-CSF was equally elicited by splenocytes from CD19Cre/IAβbstopf/f and WT mice, but not IAβbstopf/f mice (Fig. 3A).

FIGURE 3.
FIGURE 3.

Secondary CD4 T cell responses in vivo are limited when cognate interactions are restricted to B cells. (A) Ten days after s.c. immunization with MOG35–55, 2D2 CD4 T cells were restimulated with splenocytes and varying doses of MOG35–55 from IAβbstopf/f (circles), CD19Cre/IAβbstopf/f (triangles), and WT (squares) mice primed 4 d prior with i.v. CpG and MOG35–55. Cytokine production was assayed by ELISPOT. Data represent three mice per group in four experiments. For the upper panel, *p < 0.01 (IAβbstopf/f compared with WT or CD19Cre/IAβbstopf/f), **p < 0.05 (WT compared with CD19Cre/IAβbstopf/f); all other panels, *p < 0.01 compared with either other group. (B) Ten days after EAE induction, mononuclear cells from the brain and spinal cord were assessed by CD11b and CD45 expression. Data are representative of three mice per group from two experiments. (C) The number of donor cells was quantified in the spleen and CNS; *p < 0.05 when comparing WT with either IAβbstopf/f (“F”) or CD19Cre/IAβbstopf/f (“C”). Data are representative of three mice per group in two experiments. (D) Thirty days after passive EAE induction, splenocytes were isolated and cultured overnight with media (top) or MOG35–55 (bottom). IFN-γ and IL-17 production was assayed by intracellular staining. Plots are gated on CD4+Thy1.1+ cells. Data are representative of at least two mice per group in two separate experiments. (E) Seven days after transfer of encephalitogenic cells, B cell mRNA levels for IL-4, TNF-α, and IL-10 were compared with splenic B cells from naive mice. Data were representative of three mice per group. (F) B cell expression of MHCII was assessed by flow cytometry 10 d after transfer of encephalitogenic CD4 T cells. *p < 0.05 when comparing IAβbstopf/f (“F”) with either WT or CD19Cre/IAβbstopf/f (“C”). Data representative of three mice per group.

We used our in vivo mechanism for conditional expression of MHCII to test whether B cells alone are capable of coordinating CD4 T cell–mediated autoimmune neuroinflammation during passive EAE. i.v. transfer of 1 × 107 previously primed, encephalitogenic Th1 CD4 T cells (13) to WT mice resulted in 100% incidence rate of EAE, whereas IAβbstopf/f recipient mice lacking MHCII remained entirely free of disease. Similarly, CD19Cre/IAβbstopf/f recipient mice were fully resistant to clinical disease, demonstrating an insufficiency of Ag presentation by B cells to support CD4 T cell–mediated EAE (Table I).

View this table:
Table I. Clinical features of EAE in IAβbstopf/f and CD19Cre/IAβbstopf/f mice

To examine the extent of inflammation within the CNS after passive transfer of encephalitogenic CD4 T cells, CNS mononuclear cells were isolated at the peak of disease in WT mice from spinal cord and brain tissue of each mouse group. In contrast to WT CNS tissue, minimal evidence of microglial activation or leukocyte infiltration was observed in IAβbstopf/f mice (as expected) (21) or CD19Cre/IAβbstopf/f mice (Fig. 3B). At 10 d post transfer, 19.3% of mononuclear cells isolated from the CNS of WT mice were donor cells. In contrast, both IAβbstopf/f and CD19Cre/IAβbstopf/f mice had minimal accumulation (<2.5%) of donor lymphocytes within the CNS, prohibiting further characterization. Absolute numbers of donor cells paralleled this disparity (Fig. 3C). Donor CD4 T cells accumulated within the spleen of IAβbstopf/f and CD19Cre/IAβbstopf/f mice, however (Fig. 3C), and ex vivo Ag-specific recall production of IFN-γ was identical in CD19Cre/IAβbstopf/f and WT mice (Fig. 3D), serving as further evidence that B cell Ag presentation alone can elicit cognate secondary CD4 T cell responses. The cytokine profile and MHCII expression of B cells following induction of EAE did not differ between mouse groups (Fig. 3F, 3G). Thus, B cells can support Ag-specific secondary CD4 T cell responses, but have a limited capacity to propagate EAE.

CD19+CD11c+ splenocytes are one unique niche of APCs that may exert functionally distinct influences on CD4 T cells (18). The regulatory influences of these indoleamine-producing cells, mediated via CD19, are unlikely to be responsible for the lack of disease following transfer of encephalitogenic T cells, as CD19Cre/IAβbstopf/f mice homozygous for Cre expression, which eliminates CD19 expression and the indoleamine-mediated suppressive effects and is associated with heightened EAE disease severity (22), retain full resistance to EAE (Table I).

The functional relevance of B cell Ag presentation during autoimmune responses was further investigated using the frequently used active EAE model system. To avoid constraints related to homeostatic proliferation and variability associated with transfer of CD4 T cells before immunization, we reconstituted the CD4 T cell compartment of IAβbstopf/f and CD19Cre/IAβbstopf/f mice by WT thymus grafting (Supplemental Fig. 2H) (9). The results of our active EAE experiments parallel those of passive EAE, as both IAβbstopf/f and CD19Cre/IAβbstopf/f mice immunized with peptide or protein were entirely resistant to disease (Table I). To verify the ability for our system to generate sufficient levels of MHCII to support passive EAE, we used CMVCre mice, in which ubiquitous Cre expression is under the control of a human CMV minimal promoter, to rescue expression of MHCII. CMVCre/IAβbstopf/f mice have identical expression of MHCII in lymphoid tissue compared with WT mice (Supplemental Fig. 2I) and are fully susceptible to passive EAE (onset at 6.25 ± 0.25 d with maximal severity of 2.25 ± 0.72; n = 4).

Using our newly designed murine system in which subsets of APCs are capable of conditionally expressing MHCII in vivo, we have addressed the hypothesis that individual APCs contribute to CD4 T cell–mediated autoimmunity in unique ways by examining the contribution of B cells to CD4 T cell responses. Our findings are based on a knock-in model of MHCII expression rather than a transgenic approach that has been used in the past that suggested that CD4 T cell responses are not driven only by the quantity of MHCII in vivo per se, but rather the cellular source of MHCII (23). Not surprisingly, i.v. delivery of Ag, rather than s.c., was found to be optimal for stimulating initial CD4 T cell proliferation (Fig. 2), and CD19Cre/IAβbstopf/f mice are resistant to active EAE induced by s.c. immunization (Table I). Nonetheless, B cells may be relevant for s.c. Ag delivery in combination with DCs. For example, although DCs alone are sufficient to mediate peptide-induced active EAE, generation of disease after s.c. immunization with protein may require B cells (9).

Contrary to several lines of evidence suggesting an APC role for B cells during primary and secondary responses, including disease states such as EAE and multiple sclerosis, our data indicate that Ag presentation solely by B cells is not sufficient for full CD4 T cell function. However, one manner in which B cells may participate in the propagation of CD4 T cell autoreactivity is by the uptake and presentation of cognate Ag that is limited in availability (24). This may be relevant early in disease when intact myelin does not provide excess available Ag and only Ag-specific uptake of target will efficiently result in sufficient CD4 T cell activation. Another possibility involves the potential for B cells to drive unique CD4 T cell cytokine production upon restimulation after priming. Differential CD4 T cell production of GM-CSF, but not IL-17, and partial production of IFN-γ resulting from Ag-specific interactions with B cells may engender a restricted set of effector responses. Alternatively, B cells are capable of downmodulating CD4 T cell function (20, 25), and B cell–mediated tolerance may be mediated, in part, by MHCII. The cognate basis for the tolerogenic nature of B cells warrants further investigation, one that will undoubtedly be more feasible given the flexibility of our new system for conditional murine MHCII expression. Overall, comparison of CD4 T cell responses in mice expressing MHCII in specific APC lineages using this system with other cell-specific Cre mice will address the exclusive contributions by other APCs to CD4 T and greatly enhance our understanding of the pathways by which immune and autoimmune processes are generated and propagated.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the Alvin J. Siteman Cancer Center (supported in part by National Cancer Institute Cancer Center Support Grant #P30 CA91842) and the Barnes-Jewish Hospital for the use of the Embryonic Stem Cell Core, which provided all ES cell services.

Footnotes

  • This work was supported by the National Institute of Neurological Disorders and Stroke (Grant 5K08NS062138) and the Barnes-Jewish Foundation.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BM
    bone marrow
    BMDC
    bone marrow–derived dendritic cell
    DC
    dendritic cell
    EAE
    experimental autoimmune encephalomyelitis
    MHCII
    MHC class II
    WT
    wild-type.

  • Received June 22, 2012.
  • Accepted May 23, 2013.

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