Immunization with host-type CD8{alpha}+ dendritic cells reduces experimental acute GVHD in an IL-10-dependent manner

Abstract

Little is known about the role of active immunization in suppressing undesirable immune responses. Because CD8alpha(+) dendritic cells (DCs) suppress certain immune responses, we tested the hypothesis that immunization of donors with host-derived CD8alpha(+) DCs will reduce host-specific donor T-cell responses. BALB/c T cells from the animals that were immunized with B6 CD8alpha(+) DCs demonstrated, in vitro and in vivo, significantly reduced proliferation and secretion of inflammatory cytokines but showed enhanced secretion of interleukin-10 (IL-10). The responses against third-party and model antigens were preserved demonstrating antigen specificity. The in vivo relevance was further demonstrated by the reduction on graft-versus-host disease (GVHD) in both a major histocompatibility complex-mismatched clinically relevant BALB/c --> B6 model and major histocompatibility complex-matched, minor-mismatched C3H.SW --> B6 model of GVHD. Immunization of the donors that were deficient in IL-10 (IL-10(-/-)) or with CD8alpha(+) DCs from B6 class II (class II(-/-)) failed to reduce T-cell responses, demonstrating (1) a critical role for secretion of IL-10 by donor T cells and (2) a direct contact between the T cells and the CD8alpha(+) DCs. Together, these data may represent a novel strategy for reducing GVHD and suggest a broad counterintuitive role for vaccination strategies in mitigating undesirable immune responses in an antigen-specific manner.

Figure 1

Experimental design. (A) Schema of the immunization protocol. (B) Gating strategy for FACS sorting of allogeneic BM-derived DCs into subsets of CD8α⁺CD11c⁺ DCs and CD8α⁻CD11c⁺ DCs.

Figure 2

T cells from allogeneic CD8α⁺ DC–vaccinated mice. Reduced expansion and proinflammatory cytokine secretion were shown in vitro in BALB/c mice that were immunized with diluent control or allogeneic B6 BM-derived CD8α⁺ DCs or CD8α⁻ DCs as described in “Methods.” BALB/c CD90⁺ T cells (2 × 10⁵/well) were then harvested from them and cultured for 96 hours with either syngeneic or irradiated (3000 cGy) allogeneic B6 BM DCs (1 × 10⁴/well). During the final 16 hours of a 96-hour culture, cells were pulsed with [³H] thymidine and assayed for (A) proliferation. Supernatants were collected at 80 hours and assayed by for (B) IL-2, (C) IFN-γ, and (D) IL-10. Error bars represent SE. *P < .05 between diluent control and allogeneic CD8α⁺ DC–vaccinated groups. Data are from one of 3 similar experiments. *P < .05 between diluent control and allogeneic CD8α⁺ DC–vaccinated groups.

Figure 3

Immunization with allogeneic CD8α⁺ DCs reduces T-cell–proliferative response without altering their rate of apoptosis. T cells were harvested from BALB/c mice that were immunized with diluent or B6 BM-derived CD8α⁺ DC and CD8α⁻ DC subsets as described in “Methods.” BALB/c CD90⁺ T cells (2 × 10⁶/well) were then stained with CFSE and stimulated in vitro with B6 BM DCs for 96 hours and analyzed by flow cytometry. (A) Gated for fluorescein isothiocynate–conjugated CFSE and allophycocyanin-conjugated CD3⁺ T cells. Data are representative of 1 of 4 independent similar experiments. Combined data for the nonimmunized, CD8α⁺ DC–vaccinated, and CD8α⁻ DC–vaccinated are 0.77% ± 0.15%, 24.1% ± 2.4%, 4.7% ± 1.6%, and 18.1% ± 0.6%, respectively (P < .04, nonimmunized vs CD8α⁺ DC). (B) PE-conjugated annexin V and allophycocyanin-conjugated anti-CD3. P = not significant between the groups. Data are representative of 1 of 3 independent similar experiments.

Figure 4

Pretransplantation vaccination of donors with host-type CD8α⁺ DCs attenuates GVHD. Recipient B6 mice were irradiated with 1000 cGy total body irradiation (TBI) and injected with 5 × 10⁶ TCD BM and 4 × 10⁶ CD90⁺ T cells from syngeneic (squares, solid line, n = 11), allogeneic WT control (circles with dotted line, n = 12), allogeneic recipients of host-type CD8⁺ DC–vaccinated (triangles with solid line, n = 10), and CD8⁻ DC–vaccinated (diamonds with dotted line, n = 12) donors. They were evaluated for (A) survival. Error bars represent SE. *P < .05 between diluent control (red line) and host-type CD8α⁺ DC-vaccinated (triangles) groups. (B) Clinical GVHD score. Error bars represent SE. *P < .05 between controls (circles) and host-type CD8α⁺ DC–vaccinated allogeneic animals (triangles). Data are combined from 2 of 4 similar experiments. (C) Small and large intestines and livers were obtained from each group (n = 4/group) for detailed histopathologic analysis on day 7 after BMT as described in “Methods.” Coded slides were scored semiquantitatively to assess GVHD-specific pathologic damage. Total GVHD score: mean ± SE of the sum of scores for gut (small and large bowels) and livers from individual animals in each group. Error bars represent SE. *P < .05 between allorecipients of diluent control and host-type CD8α⁺ DC–vaccinated donors. (D) Recipient B6 mice were irradiated with 1000 cGy TBI and injected with 5 × 10⁶ TCD BM and 3 × 10⁶ CD90⁺ T cells from syngeneic (black solid line, n = 6), allogeneic WT control (circles with dotted line, n = 8), allogeneic recipients CD8⁺ DC-vaccinated (triangles with solid line, n = 8), and host CD8⁻ DC–vaccinated (diamonds with dotted line, n = 8) donors and evaluated for survival. Data from 2 similar experiments are combined. P < .04, CD8⁺ DCs vs control and CD8⁻ DCs.

Figure 5

Effect of host-type CD8α⁺ DC immunization on donor T-cell expansion and cytokines after allo-BMT. B6 animals were irradiated and transplanted after immunization of the donors as in “Vaccination protocol.” Splenocytes and serum were harvested from the recipients on day 7 and 14 after BMT. (A) Donor (CD45.2 or H2k^d) CD4⁺ and CD8⁺ T-cell expansion was determined by FACS analysis. Data represent the mean ± SE. *P < .05 between allorecipients of host-type CD8α⁺ DC–vaccinated donors and diluent control or host-type CD8α⁻ DC–vaccinated donors. (B-D) Serum cytokines were measured (n = 4/group) on days 7 and 14 after BMT. Serum levels of (B) IFN-γ, (C) TNF-α, and (D) IL-10. N.D indicates not detected. *P < .05 between allorecipients of host-type CD8α⁺ DC-vaccinated donors versus the diluent control. Data are from one of 2 similar experiments. (E-G) Representative figure of IL-10 production from donor T cells (n = 3 or 4/group) on day 14 after BMT (E). Gated on donor-specific (H-2k^d) cells for the double-positive population of CD3 and IL-10. The percentage IL-10⁺ donor T cells (F) and the absolute cell numbers (G). P < .05.

Figure 6

Allogeneic CD8α⁺ DC immunization preserves T-cell responses to third-party antigens. B6 donor mice were immunized with diluent or recipient BALB/c BM-derived CD8α⁺ DC or CD8α⁻ DCs as described in “Vaccination protocol.” T cells were harvested and used for in vitro and in vivo studies with C3H/HeJ recipients. (A) B6 splenic CD90⁺ T cells were stimulated with irradiated (3000 cGy) splenocytes (10⁵/well) from C3H/HeJ. During the final 6 hours of a 72-hour culture, cells were pulsed with ³H thymidine and assayed for proliferation. (B) Supernatants were collected at 66 hours and assayed by ELISA for IL-2, IFN-γ, and IL-10. Error bars represent SE. P = not significant between diluent control and BALB/c-derived CD8α⁺ DC groups. Each graph represents one of 3 similar experiments. (C) C3H/HeJ mice were lethally irradiated with 9000 cGy TBI and injected with 5 × 10⁶ TCD BM and 10⁶ CD90⁺ T cells from syngeneic (black straight line, n = 3), allogeneic diluent-treated (circles, dotted line, n = 7), or BALB/c-derived CD8⁺ DC-vaccinated (triangles, n = 6) or CD8⁻ DC-vaccinated (diamonds, n = 6) B6 animals and evaluated for survival. P = not significant between diluent control and the other groups. Data represent one of 2 similar experiments. (D) B6 OT-II transgenic mice were immunized with diluent or BALB/c BM-derived CD8α⁺ DCs or CD8α⁻ DCs as above. Responder T cells were harvested from the OT-II B6 mice and cultured for 48 hours with B6 syngeneic splenocytes that were either not pulsed or pulsed with 100nM or 500nM OVA_323-339 peptide. During the final 6 hours of a 48-hour culture, cells were pulsed with [³H] thymidine and assayed for proliferation. P = not significant between diluent control and the other immunized groups. Data are representative of one of 2 similar experiments.

Figure 7

IL-10 is required for mediating the suppressive effects of allogeneic CD8α⁺ DC vaccination. T cells from WT B6 and IL-10 KO B6 donor mice were harvested after immunization with either diluent or host BALB/c BM-derived CD8α⁺ DCs or CD8α⁻ DCs as described in “Vaccination protocol.” T cells were used as responders and stimulated with irradiated (3000 cGy) BALB/c splenocytes in an allogeneic MLR and assayed for (A) proliferation, and (B) supernatants were collected at 66 hours and assayed by ELISA for IFN-γ and IL-10. *P < .05 between T cells from WT B6 animals that were immunized with BALB/c BM-derived CD8α⁺ DC compared with diluent or CD8α⁻ DC controls. P = not significant between for T cells from IL-10 KO B6 animals in all groups. Data represent 1 of 3 similar experiments. Recipient BALB/c mice were irradiated with 8000 cGy TBI and injected with 5 × 10⁶ TCD BM and 10⁶ CD90⁺ T cells from syngeneic (black thin solid line, n = 6) or allogeneic T cells from diluent control (black bold solid line, n = 7) or BALB/c CD8⁺ DC-vaccinated (triangles, n = 8) or CD8⁻ DC-vaccinated (diamonds, n = 8) B6 WT donors or T cells from allogeneic IL-10 KO B6 donors that were immunized with diluent control (inverted triangles, n = 10), or BALB/c CD8⁺ DC-vaccinated (squares bold solid line, n = 10) or CD8⁻ DC-vaccinated (circles, dotted line, n = 9) IL-10 KO donors and evaluated for (C) survival and (D) clinical GVHD score. *P < .05 between T cells from WT B6 animals that were immunized with BALB/c BM-derived CD8α⁺ DC versus those with diluent or CD8α⁻ DC controls. P = not significant between for T cells from IL-10 KO B6 animals in all groups. Data represent 1 of 3 similar experiments. Data are from 2 combined experiments with similar results.

Figure 8

Regulation of T cells is dependent on contact with the infused allogeneic CD8α⁺ DCs. BALB/c mice were vaccinated with B6 H2-Ab1^−/− BM-derived CD8α⁺ DCs or CD8α⁻ DCs as in “Vaccination protocol.” T cells were harvested from these BALB/c animals and cultured for 72 hours with syngeneic or WT B6 allogeneic irradiated (3000 cGy) splenocytes and analyzed for proliferation after [³H] thymidine incorporation. P = not significant between the groups. Data are from one of 2 similar experiments.