A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent but CD70-dependent mechanism in vivo

Abstract

Interferon (IFN)-gamma, a cytokine critical for resistance to infection and tumors, is produced by CD4(+) helper T lymphocytes after stimulation by cultured dendritic cells (DCs) that secrete a cofactor, interleukin (IL)-12. We have identified a major IL-12-independent pathway whereby DCs induce IFN-gamma-secreting T helper (Th)1 CD4(+) T cells in vivo. This pathway requires the membrane-associated tumor necrosis family member CD70 and was identified by targeting the LACK antigen from Leishmania major within an antibody to CD205 (DEC-205), an uptake receptor on a subset of DCs. Another major DC subset, targeted with 33D1 anti-DCIR2 antibody, also induced IFN-gamma in vivo but required IL-12, not CD70. Isolated CD205(+) DCs expressed cell surface CD70 when presenting antigen to T cell receptor transgenic T cells, and this distinction was independent of maturation stimuli. CD70 was also essential for CD205(+) DC function in vivo. Detection of the IL-12-independent IFN-gamma pathway was obscured with nontargeted LACK, which was presented by both DC subsets. This in situ analysis points to CD70 as a decision maker for Th1 differentiation by CD205(+) DCs, even in Th2-prone BALB/c animals and potentially in vaccine design. The results indicate that two DC subsets have innate propensities to differentially affect the Th1/Th2 balance in vivo and by distinct mechanisms.

Figure 1.

αDEC and 33D1 mAbs target LACK in vivo to distinct DC subsets. (A) BALB/c mice (five per condition) were immunized i.p. with αDEC-LACK, 33D1-LACK, LACK, and Ig-LACK in the presence of αCD40 and poly IC, or with PBS. 12 h later, splenocytes were enriched for CD11c⁺ cells by MACS⁺ selection and separated into DEC⁺ and DEC⁻ subsets (see Materials and methods). The DCs were added to thy-1.1⁺ CFSE-labeled, LACK-specific TCR transgenic T cells, and proliferation was assessed by CFSE dilution 3.5 d later. Representative of three experiments. (B) 3 × 10⁶ CFSE-labeled TCR transgenic cells as in A were administered i.v. to thy-1.2⁺ mice 1 d before immunization i.p. with graded doses of αDEC-LACK, 33D1-LACK, Ig-LACK, or LACK protein. Spleens were harvested at 3 d to enumerate thy-1.1⁺ T cells that had diluted CFSE. The expansion of transgenic T cells is displayed as total cell numbers on the left, and representative CFSE dilution plots, gated on thy-1.1⁺ LACK transgenic CD4⁺ T cells in response to the indicated doses of fusion mAb or LACK protein, are on the right.

Figure 2.

Contrasting cytokine production by T cells immunized with different forms of LACK. (A) BALB/c mice were injected i.p. with PBS, 10 μg αDEC-LACK, 10 μg 33D1-LACK, 10 μg control Ig-LACK, or 30 μg LACK in the presence of αCD40 and poly IC. 3 wk later, splenocytes were enriched for CD4⁺ cells (MACS selection) and restimulated with CD11c⁺ cells plus LACK-dominant peptide. 2 d later, the numbers of IFN-γ– (left) and IL-4– (right) producing cells were revealed by ELISPOT. Three experiments with two mice per group. (B) As in A, but mice were both primed and boosted at 3 wk with different forms of LACK. 6 d later, anti-LACK–specific antibody titers and isotypes were quantified by ELISA. Each symbol is an individual experiment. (C) 3 × 10⁶ CFSE-labeled, thy-1.1⁺ LACK-specific TCR transgenic cells were transferred i.v. into thy-1.2⁺ BALB/c mice. 1 d later, the mice were immunized i.p. with the different LACK antigen forms in the presence of αCD40 and poly IC, 10⁶ L. major metacyclic promastigotes, or PBS. 6 d later, the priming of LACK-specific transgenic T cells was assessed for intracellular IFN-γ production by gating on thy-1.1⁺ splenocytes (left), or for IL-4 production by ELISPOT of positively selected thy-1.1⁺ splenocytes restimulated for 4 h in the presence of PMA and ionomycin (right). Representative of three experiments.

Figure 3.

αDEC-LACK induces IFN-γ secretion by an IL-12–independent pathway. (A) IL-12p40^−/− mice and control littermates were immunized i.p. as indicated in Fig. 2 and on the x axis. 3 wk later, the frequency of IFN-γ⁺ CD4⁺ CD3⁺ splenic T cells was determined by intracellular staining (left), or CD4⁺ splenocytes were restimulated in vitro with CD11c⁺ cells, medium, or LACK peptide for 36–48 h to measure IL-4– secreting cells by ELISPOT (right). (B) As in A, but each row shows a different dose of the different forms of LACK antigen. (C) As in A, but WT and IL-12 KO mice were immunized with P. yoelli circumsporozoite protein (CSP), either 10 μg DEC-CSP, 10 μg 33D1-CSP, and 10 μg Ig-CSP in the presence of αCD40 and poly IC, or PBS. Representative of three, three, and two individual experiments with two pooled mice per condition, respectively.

Figure 4.

DEC⁻ DCs differentiate Th2 type CD4⁺ T cells but prime IFN-γ secretion with IL-12–bearing DEC⁺ DCs. (A) BALB/c mice were immunized i.p. as indicated in Fig. 2 and the right inset for 10–12 h. DEC⁺ and DEC⁻ subsets were sorted from spleens and added for 3.5 d to thy-1.1⁺ CFSE-labeled, LACK-specific TCR transgenic cells. IL-2, IFN-γ, IL-4, and IL-5 production were measured in the supernatants by ELISA. The symbols represent individual experiments with five mice per condition, and the horizontal bars represent the mean between the different experiments. (B) As in A, but the DC subsets from either WT (shown) or IL-12^−/− mice (see Fig. S6) were used to stimulate T cell proliferation (CFSE dilution, x axis) as well as IFN-γ production (y axis; monensin was added overnight in the absence of any restimulation at day 3). (C) DEC⁻ DCs were isolated from mice given 33D1-LACK, LACK in the presence of αCD40 and poly IC, or PBS 12 h earlier (same cells as B), but we then added an equal number of DCs from WT (WT) and IL-12p40^−/− mice as indicated on the x axis. The donor mice had been given anti-CD40 and poly IC, but no LACK antigen, 12 h beforehand (mature), or just PBS. As in B, T cell proliferation and IFN-γ production were monitored. One of two similar experiments.

Figure 5.

CD70 mediates the stimulatory function of DEC⁺ DCs, but not DEC⁻ DCs. (A) Mice were immunized i.p. as in Fig. 2 and as shown at the top for 10–12 h. Sorted DEC⁺ and DEC⁻ subsets were added for 1 d to thy-1.1⁺ CFSE-labeled, LACK-specific TCR transgenic cells, and the CD11c⁺ DCs (the only CD70⁺ cells in the cultures) were monitored by FACS for reactivity to αCD70 (dotted line) or isotype control mAb (gray). (B) As in A, but the cells were cultured for 3.5 d in the presence of αCD70 or control IgG2b (1 μg/ml). Inhibition of T cell growth, assessed by CFSE dilution, is noted with arrowhead and arrows. (C) As in B, but BALB/c mice were injected with αDEC-LACK alone and in the presence of αCD40, poly IC, or both. IFN-γ production by proliferating LACK transgenic cells was determined by intracellular cytokine staining. (D) As in B, but T cell activation was monitored by CD69 staining at days 1.5 and 3.5. (E) As in B, but αCD70 was added 1 d after co-culture. B–E are representative of four, two, five, and two experiments, respectively, with five mice per condition.

Figure 6.

Contributions of CD70 to IFN-γ secretion in response to DEC⁺ DCs in vivo. BALB/c mice were injected i.p. with 50 μg αCD70 mAb 1 d before i.p. immunization with 10 μg DEC-LACK, 10 μg 33D1-LACK, 50 μg LACK protein, and 10 μg Ig-LACK in the presence of αCD40 and poly IC, or PBS. 3 wk later, the frequency of IFN-γ⁺ CD4⁺ CD3⁺ splenic T cells was determined by intracellular staining. One of two similar experiments.