Murine CD4+ T Cell Responses Are Inhibited by Cytotoxic T Cell-Mediated Killing of Dendritic Cells and Are Restored by Antigen Transfer

Cytotoxic T lymphocytes (CTL) provide protection against pathogens and tumors. In addition, experiments in mouse models have shown that CTL can also kill antigen-presenting dendritic cells (DC), reducing their ability to activate primary and secondary CD8+ T cell responses. In contrast, the effects of CTL-mediated killing on CD4+ T cell responses have not been fully investigated. Here we use adoptive transfer of TCR transgenic T cells and DC immunization to show that specific CTL significantly inhibited CD4+ T cell proliferation induced by DC loaded with peptide or low concentrations of protein antigen. In contrast, CTL had little effect on CD4+ T cell proliferation induced by DC loaded with high protein concentrations or expressing antigen endogenously, even if these DC were efficiently killed and failed to accumulate in the lymph node (LN). Residual CD4+ T cell proliferation was due to the transfer of antigen from carrier DC to host APC, and predominantly involved skin DC populations. Importantly, the proliferating CD4+ T cells also developed into IFN-γ producing memory cells, a property normally requiring direct presentation by activated DC. Thus, CTL-mediated DC killing can inhibit CD4+ T cell proliferation, with the extent of inhibition being determined by the form and amount of antigen used to load DC. In the presence of high antigen concentrations, antigen transfer to host DC enables the generation of CD4+ T cell responses regardless of DC killing, and suggests mechanisms whereby CD4+ T cell responses can be amplified

Antigen transfer from injected DC to host DC occurs at the site of DC injection.

<p>(A) C57BL/6 mice were injected with CD45.1⁺ WT DC or CD45.1⁺ WT DC loaded with OVA protein (2 mg/ml). The dLN were collected 24 h later, and the CD8⁺ DC, the CD8⁻CD205⁺ skin-derived DC and the CD8⁻CD205⁻ double negative DC populations were sorted and cultured in duplicate with CFSE-labelled, purified OT-I or OT-II T for 3 days or 5 days, respectively. OVA-specific proliferation was evaluated as CFSE dilution by flow cytometry. Each symbol represents the mean+SEM of the percentage of divided cells/well. Combined data from two independent experiments that gave similar results are shown. (B) C57BL/6 were injected with CFSE-labelled CD45-congenic OT-II CD4⁺ T cells and immunized 24 h later with WT or MHCII^−/− DC that had been loaded with OVA protein (2 mg/ml), and treated with Ptx or left untreated. CD4⁺ T cell proliferation in dLN was determined by flow cytometry 3 days after DC immunization. Representative flow cytometry histograms of CD45.1⁺CD4⁺ T cells from individual dLN are shown on the left. The mean ± SEM of the percent divided cells in each group is shown. The bar graph on the right shows mean+SEM of the number of divided CD45.1⁺CD4⁺ T cells/dLN. Combined results from two independent experiments each with 5 mice per group are shown.</p

Host DC capture antigen from injected DC and present to CD4⁺ T cells in vitro.

<p>A) C57BL/6 mice were immunized with CD45.1⁺ DC loaded with DQ-OVA or no OVA. At 24 h after DC injection, CD45.1⁻CD45.2⁺ host cells in the dLN were identified and examined for CD11c expression and DQ-OVA uptake by flow cytometry. Representative flow cytometry dot plots of live LN cells are shown on the left and CD45.2⁺ cells are shown on the right. The number of events in each gate is shown. (B) C57BL/6 mice were injected with CD45.1⁺ DC, or CD45.1⁺ DC loaded with OVA protein (2 mg/ml). The dLN were collected 24 h later, and the total DC population (donor and host) and CD45.1⁻ DC population (host only) were magnetically purified and cultured in triplicate with CFSE-labelled, purified OT-I or OT-II T for 3 days or 5 days, respectively. OVA-specific proliferation was evaluated as CFSE dilution by flow cytometry. Each symbol shows mean+SEM of the percentage of divided cells/well. Combined data from two independent experiments that gave similar results are shown. (C) As in B, except that some mice were injected i.v. with OT-I CTL 1 day before DC transfer, and only host DC were tested. Symbols shows mean+SEM of the percentage of divided cells/well. Combined data from two independent experiments that gave similar results are shown.</p

Induction of IFN-γ production by CD4⁺ T is not prevented by CTL-mediated DC killing.

<p>C57BL/6 mice were injected with CD45-congenic OT-II CD4⁺ T cells; OT-I CTL were injected in some of the mice at the same time. 24 h later, mice were immunized s.c. with DC loaded with different forms of OVA. 19 days after immunization, spleens were collected and the total number of OVA-specific, IFN-γ-producing CD4⁺ T cells was determined by intracellular staining. Bar graphs show mean+SEM. (A) Mice were immunized with WT or MHCII^−/− DC loaded with OVA protein (2 mg/ml). The bar graph shows data from one of two similar experiments, each with 5 mice per group, that gave similar results. (B) Mice were immunized with OVAtg DC. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037481#s2" target="_blank">Results</a> are from 5 mice per group. (C) Mice were immunized with WT or MHCII^−/− DC loaded with OVA protein (2 mg/ml); some of the DC were also treated with Ptx. The bar graph shows data from 5 mice per group.</p