Enhancement of tumour-specific immune responses in vivo by 'MHC loading-enhancer' (MLE)

Abstract

Background: Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered.

Principal findings: Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with 'MHC-loading enhancer' (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE.

Conclusion: MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy.

Figure 1. Influence of MLE on the class II MHC peptide-loading of dendritic cells.

(A) Cell surface loading. HLA-DR4 expressing dendritic cells (DC) generated from the bone marrow of HLA-DR4 transgenic mice were incubated for 4 h with medium alone (left panel) or with 5 µg/ml biotinylated HA 306–318 peptide in the absence (middle panel) or presence of 250 µM AdEtOH, the model MLE compound used throughout this study (right panel). Contour plots are shown for DC after staining with anti-HLA-DR antibody (→ MHC expression) and streptavidin (→ peptide load). Mean peptide loading (MFI of streptavidin signal) is indicated. (B) CD4+ T cell response. DC from HLA-DR4tg mice (left panel) and from HLA-DR1tg mice (right panel) were pulsed for 4 h with indicated amounts of HA 306–318 peptide in the absence (open circle) and presence (closed circle) of 250 µM AdEtOH. The cells were used to challenge HA 306–318 specific, HLA-DR4-restricted 8475/94 cells and HLA-DR1-restricted EvHA/X5 T cell hybridoma cells, respectively. Background proliferation was measured in absence of peptide (dashed line).

Figure 2. Selective enhancement of CD4+ T cell responses in vivo by MLE compounds.

(A) Cell surface loading of MLE-susceptible and –non-susceptible MHC molecules. DC generated from non-susceptible BALB/c mice (left panel) and AdEtOH-susceptible HLA-DR1tg (right panel) were incubated for 4 h with the indicated amounts of biotinylated HA 306–318 peptide in the absence (open circles) or presence (closed circles) of 250 µM AdEtOH. Peptide loading was determined on CD11c+ cells by analyzing the mean fluorescence of the streptavidin-signal gated on a distinct expression of HLA-DR. Background fluorescence was detected in the absence of biotinylated peptide (dashed line). (B) Peptide priming of mice. BALB/c (upper panel) and DR1tg (lower panel) mice were s.c. primed with 100 µg and 3 µg HA 306–318, respectively in IFA/CpG supplemented without (left panel) or with AdEtOH (middle panel). Specific T cell response was determined by intracellular flow cytometry staining at day 12 after priming. Lymph node cells were incubated for 6 hrs in the presence of 10 µg HA 306–318 or 10 µg MOG 35–55 as irrelevant control peptide (right panel) and αCD28 antibody. 3 µg/ml Brefeldin A was added for the last 2 h. Intracellular IFNγ-production was analyzed on CD4+ CD154+ double positive T cells. Numbers indicate frequency of CD4+ CD154+ IFNγ+ cells among total CD4+ cells. Data representative of at least two independent experiments are shown.

Figure 3. Vaccination in presence of MLE increases the number of antigen-specific IFNγ-producing T cells.

(A) Determination of IFNγ in an Elispot assay. 12 days after vaccination, 1×10⁶ lymph node cells from mice primed with 3 µg HA 306–318 in IFA/CpG supplemented without (left panel) or with AdEtOH (right panel) were incubated with titrated amounts of HA306–318 peptide on a plate coated with α-IFNγ antibody. Detection was carried out 48 hrs later by determining the spot number in each well. Spots represent single IFNγ+ cells. (B) Statistical analysis of the T cell response. Summarized Elispot data obtained from groups of BALB/c mice (left panel, n = 10) and HLA-DR1tg mice (right panel, n = 9) were analyzed using student's t test.

Figure 4. MLE enhances the T cell response against NY-ESO-1 derived epitopes.

(A) Cell surface loading of NY-ESO-1 epitopes. L929 fibroblasts transfected with HLA-DR1 (left panel) or HLA-DR4 (right panel) were incubated for 4 h with titrated amounts of NY-ESO-1 89–101 or NY-ESO-1 119–143, respectively. Loading was performed in the absence (closed circles) or presence (open circles) of 250 µM AdEtOH. Non-transfected L929 cells were used as a negative control (left side). Peptide loading was determined by analyzing the mean fluorescence of the streptavidin-signal gated on a distinct expression of HLA-DR. Background fluorescence was detected in the absence of biotinylated peptide (dashed line). (B) Detection of tumour-specific T cell response in vivo. Groups of HLA-DR1tg (left panel, n = 13) or HLA-DR4tg (right panel, n = 10) mice were s.c. primed with 5 µg of the respective NY-ESO-1 epitopes in IFA/CpG supplemented with or without AdEtOH. 12 days after vaccination, 1×10⁶ Lymph node cells were incubated with titrated amounts of NY-ESO-1 89–101 or NY-ESO-1 119–143, respectively. IFNγ-detection was carried out 48 hrs later using an Elispot assay and summarized data were analyzed using student's t test.

Figure 5. MLE enhance the T cell response against NY-ESO-1 protein.

(A) Determination of T cell response by intracellular cytokine staining. HLA-DR1tg mice were s.c. primed with 10 µg NY-ESO-1 protein in IFA/CpG supplemented with (right panel) or without AdEtOH (middle panel). The specific T cell response was determined by intracellular flow cytometry staining 12 days after priming. Lymph node cells were restimulated for 6 h in the presence of 10 µg NY-ESO-1 protein or control peptide (left panel) together with αCD28 antibody. 3 µg/ml Brefeldin A was added for the last 2 h. Intracellular IFNγ-production was analyzed on CD4+ CD154+ double positive T cells. Numbers indicate frequency of CD4+ CD154+ IFNγ+ cells among total CD4+ cells. Data is representative of two independent experiments. (B) Dose-response. Groups of 5 mice were primed with 10 µg NY-ESO-1 protein emulsified in IFA/CpG supplemented with (open circle) or without AdEtOH (closed circle). 12 days after vaccination 1×10⁶ Lymph node cells were ex vivo challenged with titrated amounts of NY-ESO-1 protein and IFNγ-detection was carried out 48 hrs later using an Elispot assay. Spot numbers represent single IFNγ+ cells.