Cutting edge: Conditional MHC class II expression reveals a limited role for B cell antigen presentation in primary and secondary CD4 T cell responses

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.

Figure 1

MHCII expression is functionally abrogated in IA_β^bstop^f/f mice and restricted to B cells in CD19^Cre/IA_β^bstop^f/f mice. MHCII expression was analyzed after crossing CD19^Cre mice (17) to IA_β^bstop^f/f mice. (A) BM (top) and spleen (bottom) expression of MHCII in IA_β^bstop^f/f (shaded), CD19^Cre/IA_β^bstop^f/f (blue line) and WT (black line) mice. Histograms are from singlet cells gated on CD11c+CD19− cells (left), and CD19^int (middle) and CD19^hi populations. Data representative of six or more mice per group analyzed in three experiments. (B) BMDC generated from IA_β^bstop^f/f (gray), CD19^Cre/IA_β^bstop^f/f (white) and WT (black) mice were pulsed with MOG_35-55 peptide or rMOG protein then co-cultured 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) MOG_35-55 was added to spleens from WT (black), IA_β^bstop^f/f (red), and CD19^Cre/IA_β^bstop^f/f (blue) mice before (square) and after (circle) depletion of B cells. Further enrichment for DCs was also performed (X). CTLL-2 were incubated with supernatants from MOG.15 and splenic APC cultures with varying doses of MOG_35-55. Data are representative of triplicate samples in two separate experiments.

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 IA_β^bstop^f/f (“F”; red), CD19^Cre/IA_β^bstop^f/f (“C”; blue) and WT (black) mice one day prior to immunization with 50 μg MOG_35-55 i.v. (A) or 150 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 three days after immunization. Percent of dividing 2D2 cells are shown for i.v. peptide (C) or protein (D), or SQ MOG_35-55 (E) or rMOG SQ (E) in CFA. (G) Percent donor 2D2 cells isolated from mice immunized with SQ or i.v. MOG_35-55, or SQ or i.v. rMOG, with CD62L^low expression. Each graph represents at least three mice per group. *p <0.001 (C–F) or p<0.01 (G) in comparison to either other group; **p <0.01 in comparison to WT (C&D) or IA_β^bstop^f/f (G).

Figure 3

Secondary CD4 T cell responses in vivo are limited when cognate interactions are restricted to B cells. (A) Ten days after SQ immunization with MOG_35-55, 2D2 CD4 T cells were re-stimulated with splenocytes and varying doses of MOG_35-55 from IA_β^bstop^f/f (circles), CD19^Cre/IA_β^bstop^f/f (triangles) and WT (squares) mice primed four days prior with i.v. CpG and MOG_35-55. Cytokine production was assayed by ELISPOT. Data represent three mice per group in four experiments. For the upper panel, *p<0.01 (IA_β^bstop^f/f compared with WT or CD19^Cre/IA_β^bstop^f/f), **p<0.05 (WT compared with CD19^Cre/IA_β^bstop^f/f); all other panels, *p<0.01 compared to either other group. (B) Ten days following EAE induction, mononuclear cells from the brain and spinal cord were assessed by CD11b and CD45 expression. Data 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 to either IA_β^bstop^f/f (“F”) or CD19^Cre/IA_β^bstop^f/f (“C”). Data representative of three mice per group in two experiments. (D) 30 days after passive EAE induction, splenocytes were isolated and cultured overnight with media (top) or MOG_35-55 (bottom). IFN-γ and IL-17 production was assayed by intracellular staining. Plots are gated on CD4+Thy1.1+ cells. Data 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 was compared to splenic B cells from naïve mice. Data representative of three mice per group. (F) B cell expression of MHCII was assessed by flow cytometry ten days following transfer of encephalitogenic CD4 T cells. *p<0.05 when comparing IA_β^bstop^f/f (“F”) to either WT or CD19^Cre/IA_β^bstop^f/f (“C”). Data representative of three mice per group.