Differential contribution of dendritic cell CD1d to NKT cell-enhanced humoral immunity and CD8+ T cell activation

Abstract

CD1d-restricted type I NKT cells provide help for specific antibody production. B cells, which have captured and presented a T-dependent, antigen-derived peptide on MHC class II and CD1d-binding glycolipid α-GC on CD1d, respectively, activate Th and NKT cells to elicit B cell help. However, the role of the DC CD1d in humoral immunity remains unknown. We therefore constructed mixed bone marrow chimeras containing CD1d-expressing, DTR-transgenic DCs and CD1d(+) or CD1d(-) nontransgenic DCs. Following DT-mediated DC ablation and immunization, we observed that the primary and secondary antibody responses were equivalent in the presence of CD1d(+) and CD1d(-) DCs. In contrast, a total ablation of DCs delayed the primary antibody response. Further experiments revealed that depletion of CD1d(+) DCs blocked in vivo expansion of antigen-specific cytotoxic (CD8(+)) T lymphocytes. These results provide a clear demonstration that although CD1d expression on DCs is essential for NKT-enhanced CD8(+) T cell expansion, it is dispensable for specific antibody production.

Figure 1.. Bone marrow chimera generation and characterization.

(A) Outline of the strategy used for generating bone marrow chimeras. Recipient mice (CD45.1⁺) were lethally irradiated and engrafted with a 50/50 mixture or 100% of bone marrow cells from donor mice (CD45.2⁺). (B) After 12 weeks, splenocytes were obtained from chimeric mice and analyzed by flow cytometry for reconstitution of CD45.2⁺ donor cells. Density plots show reconstitution of CD45.2⁺ cells in all three chimeras (upper panel). Histogram overlay (lower panel) shows staining of cell-surface CD1d on total splenocytes (black line) versus background staining with an isotype control mAb (gray shaded). The graph on the right shows consistent engraftment of CD45.2⁺ donor cells for five mice/group. (C and D) Density plots show the effect of vehicle (upper panels) and DT (lower panels) on the frequency of (C) CD11c⁺GFP⁺ and (D) CD11c⁺CD1d⁺ DCs in splenocytes. The graph on the right shows the effect of DT on frequency of splenic CD11c⁺/GFP⁺ DTR-transgenic cells for each chimera (mean±sem for five mice/group). Statistically significant differences among experimental groups are indicated by asterisks.

Figure 2.. DT treatment does not deplete NKT cells.

Splenocytes were obtained from vehicle or DT-treated (A) donor DTR mice and (B) reconstituted chimeras and then assessed by flow cytometry. (A) Dot-plots in the upper row show CD1d tetramer binding versus expression of the GFP/DTR transgene. The lower row shows expression of TCR-β versus binding of the CD1d tetramer. (B) Expression of TCR-β versus binding of the CD1d tetramer for the chimeras used in this study. Left and right columns depict samples from vehicle and DT-treated mice, respectively. (C) Graph indicates the effect of DT treatment on NKT frequency in the chimeras for three mice/group.

Figure 3.. CD1d expression by DCs is dispensable for NKT-enhanced, specific antibody production.

All three chimeras (C57BL/6:DTR, CD1d^−/−:DTR, 100% DTR) were divided in two groups: receiving vehicle (black bars; n=9) or DT (gray bars; n=10) and immunized with NP-KLH plus α-GC, as described in Materials and Methods. Anti-NP IgG1 endpoint titers were measured by ELISA, as described previously [10, 11]. Data show endpoint titers ± sd. (A, C, and E) Graphs show primary antibody responses measured at 7, 14, and 21 days after immunization. (B, D, and F) Graphs show recall responses measured 31 and 35 days after immunization. (G and H) Graphs show response to NP-KLH immunization only in each of the 100% DTR chimera. Similar results were obtained in the mixed chimeras (not depicted). (I) Graph shows the number of CD11c⁺/GFP⁺ cells detectable in the peripheral blood of mice used in this experiment. Data show mean ± sem for four mice/group. Asterisks indicate significant differences in cell frequency as compared to pre-treatment (day 0).

Figure 4.. CD1d expression by DCs is required for NKT-enhanced CD8⁺ T cell expansion.

Twenty-four hours after vehicle or DT treatment, CFSE-labeled CD8⁺OT-1 cells were transferred into (A) CD11c-DTR mice or (B) 100% DTR chimeras. After a further 24 h, recipient mice were immunized i.p. with OVA plus α-GC (n=3), OVA alone (n=3), or HEL plus α-GC (n=3). After 72 h, splenocytes were analyzed by flow cytometry. After initial gating on FSC⁺CD8⁺ cells, OVA-H2-K^b Pentamer⁺CD19⁻ cells (density plots) were analyzed for CFSE dilution (histograms). OVA-specific CD8⁺ T cell response in (A) CD11c-DTR mice, (B) 100% DTR chimeras, or (C) CD1d^−/−:DTR chimeras treated with vehicle (upper panels) and DT (lower panels) is shown as CFSE dilution after 72 h. The bar graphs on the right summarize the results from two independent experiments. Asterisks indicate statistically significant differences between experimental groups.