Influence of CD4 T cells and the source of major histocompatibility complex class II-restricted peptides on cytotoxic T-cell priming by dendritic cells

Abstract

We have previously reported that bone marrow derived dendritic cells (DC) pulsed with major histocompatibility complex (MHC) class I-restricted peptide efficiently prime a cytotoxic T lymphocyte (CTL) response in vivo. Here we assess the involvement of CD4(+) T cells in the induction of CD8(+) CTL by DC by testing the ability of class II-deficient (C2D) DC, class II mutant (Alpha beta mut) DC and autologous serum generated DC (AS DC) to present class I-restricted antigens in vitro and in vivo. DC generated from the bone marrow of class II knockout mice and transgenic mice expressing a mutant class II that can not bind CD4 were phenotypically similar to wild type (wt) DC, except with regard to MHC class II expression. The C2D and Alpha beta mut DC, though fully capable of presenting the class I-restricted ovalbumin (OVA) peptide to a T-cell hybridoma in vitro, failed to prime a CTL response in vivo. Restoration of class II expression on C2D DC allowed priming of a CTL response; thus, the defect in CTL priming was indeed caused by the absence of class II expression. Likewise, DC generated in autologous serum were unable to prime a CTL response as these DC only express 'self' class II epitopes and therefore would not activate syngeneic CD4(+) T cells. Addition of exogenous class II epitopes rescued the ability of AS DC to prime a CTL response. These observations provide convincing evidence that efficient CTL induction by DC in vivo requires concomitant presentation of class II epitopes for CD4(+) T-cell induction.

Figure 1

Phenotypic analysis of C2D, Aβmut, Aβwt and wt DC. Mature DC generated in FCS from wt (C57BL/6 or Aβwt), C2D, or Aβmut mice were stained with FITC-labelled mAb specific for the indicated cell surface molecule as described in Materials and Methods then analysed. Representative data is shown.

Figure 2

In vitro presentation of class I- and class II-restricted OVA peptide to OVA-specific T-cell hybridomas by C2D and Aβmut DC. Mature day 10 DC generated from wt (C57BL/6 or Aβwt), C2D or Aβmut mice in RPMI with 5% FCS were pulsed with OVA_257–264 class I peptide (a) or OVA_258–276 class II peptide (b) as described in Materials and Methods. DC were washed with PBS and incubated with H-2K^b + OVA I peptide-specific RF33.70 T-cell hybridomas (a) or I-A^b + OVA II peptide-specific MF2.2D9 T cell hybridomas (b). T cell stimulation was determined by measuring the amount of IL-2 in the supernatant by ELISA. Representative data is shown.

Figure 3

Class II deficient and class II mutant DC can not prime a CTL response in vivo. Aβwt mice were immunized with 10⁵ DC which were either pulsed with 10 ng/ml OVA_257–264 class I peptide or unpulsed. Seven days post-immunization, non-adherent splenocytes were restimulated as described in materials and methods. After 5 days of restimulation, effectors were harvested and cytolytic activity was measured against peptide pulsed RMA-S target cells in a 4-hr ⁵¹Cr release assay. Similar results were obtained against EG7.OVA and EL4 targets (data not shown).

Figure 4

Phenotypic analysis of AS DC and FCS DC. Mature DC generated in AS or FCS from wt C57BL/6 mice were stained with FITC-labelled mAb specific for the indicated cell surface molecule as described in materials and methods then analysed. Representative data is shown.

Figure 5

In vitro presentation of class I- and class II-restricted OVA peptide to OVA-specific T-cell hybridomas by AS and FCS DC. Mature day 10 DC generated from wt C57BL/6 mice in media containing FCS or pooled AS were pulsed with OVA_257–264 class I peptide (a) or OVA_258–276 class II peptide (b) as described in Materials and Methods. DC were washed with PBS and incubated with H-2K^b + OVA I peptide-specific RF33.70 T-cell hybridomas (a) or I-A^b + OVA II peptide-specific MF2.2D9 T-cell hybridomas (b). T-cell stimulation was determined by measuring the amount of IL-2 in the supernatant by ELISA. Representative data is shown.

Figure 6

DC generated in AS pulsed with the class I OVA peptide can not prime CTL in vivo unless a source of class II antigen is added. DC were generated in FCS or pooled AS from C57BL/6 mice. On day 7 of culture 5 mg/ml of BSA or OVA protein was added to some of the AS DC cultures. Mature day 10 DC were pulsed with 0·05 µg/ml of either the OVA or Mut-1 H-2K^b-resticted peptide (AS DC pulsed with OVA on day 7 were not pulsed with peptide). DC were washed in PBS and 10⁵ DC were injected i.v. into naive mice. Seven days post-immunization, non-adherent splenocytes were restimulated as described in Materials and Methods. BSA or OVA was added to the restimulation media of those groups. After 5 days, CTL activity was measured against specific and non-specific targets in a 4-hr 51Cr-release assay. The mean and standard deviation of triplicate samples are plotted. Similar results were observed on EL4 and EG7.OVA targets (data not shown).

Figure 7

In vivo CTL activity induced by AS DC presenting class II antigen is reduced in CD4-depleted mice. DC were generated in FCS or pooled AS from C57BL/6 mice. On day 8 of culture, 5 mg/ml of BSA was added to some of the AS DC cultures. Mature day 10 DC were pulsed overnight in culture with 0·05 µg/ml of either the OVA or Mut-1 H-2K^b-resticted peptide. DC were washed in PBS and 10⁵ DC were injected i.v. into non-depleted or CD4-depleted mice. Eight days post-immunization, non-adherent splenocytes were restimulated as described in Materials and Methods. BSA was added to the restimulation media of those groups where BSA was used for pulsing DC. After 5 days, CTL activity was measured against specific and non-specific targets in a 4-hr ⁵¹Cr-release assay. The mean and standard deviation of triplicate samples are plotted.