Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens

Abstract

In the control of T-helper type I (Th-1) polarization, dendritic cells (DCs) must interpret a complex array of stimuli, many of which are poorly understood. Here we demonstrate that Th-1 polarization is heavily influenced by DC-autonomous phenomena triggered by the loading of DCs with antigenically matched major histocompatibility complex (MHC) class I and class II determinants, that is, class I and II peptide epitopes exhibiting significant amino acid sequence overlap (such as would be physiologically present during infectious processes requiring Th-1 immunity for clearance). Data were derived from 13 independent antigenic models including whole-cell systems, single-protein systems, and 3 different pairs of overlapping class I and II binding epitopes. Once loaded with matched class I and II antigens, these "Th-1 DCs" exhibited differential cytokine secretion and surface marker expression, a distinct transcriptional signature, and acquired the ability to enhance generation of CD8(+) T lymphocytes. Mechanistically, tRNA-synthetases were implicated as components of a putative sensor complex involved in the comparison of class I and II epitopes. These data provide rigorous conceptual explanations for the process of Th-1 polarization and the antigenic specificity of cognate T-cell help, enhance the understanding of Th-1 responses, and should contribute to the formulation of more effective vaccination strategies.

Figure 1

DC cultures are devoid of accessory CD3⁺ cells. (A) Isotype control. (B) DC culture on day 8 demonstrating no CD3⁺ events. (C) CD3⁺ control. x-axis, CD209-PE; y-axis, CD3-PerCP.

Figure 2

DCs loaded with matched class I and II antigens exhibit enhanced IL-12 secretion and CD83 expression. (A) Double-loading of DCs with matched MHC class I and II determinants enhances DC IL-12 secretion in a single-antigen system. equals IL-12 secretion from DCs loaded with matched class I (plasmid) and class II (soluble protein). □ equals IL-12 secretion from other DC loaded by any other method. y-axis: pg IL-12/mL/10⁶ cells (A,B,D). (B) Double-loading of DCs with matched MHC class I and II antigens enhances DC IL-12 secretion. Eight experiments shown (double unmatched, 5 experiments shown). (C) Double-loading of DC with matched class I and II determinants causes enhanced up-regulation of CD83 expression. Average MFI value of unloaded and singly loaded DCs equals 0. □ equals percent by which CD83 MFI of all unloaded or singly loaded DCs differs from the average. equals percent by which CD83 MFI of all doubly loaded DCs differs from the average (P < .001). y-axis equals percent difference of CD83 MFI of CD83⁺ DC from the average of unloaded and singly loaded DCs. (D) Loading of DC MHC class I by electroporation of rHA and class II by incubation of rHA enhances DC IL-12 secretion. equals IL-12 secretion from DCs loaded with matched class I and class II proteins (rHA). □ equals IL-12 secretion from DC loaded singly or loaded doubly with disparate class I and class II antigens (ie, rHA and luciferase).

Figure 3

Differential regulation of CD83, CD40, and CTLA-4 is dependent upon the double-loading of DCs with matched class I and II antigens. (A) Percent difference of CD83 expression from average of unloaded and singly loaded controls between matched doubly loaded () and unmatched doubly loaded (□) DCs (P = .009). y-axis equals percent difference from average of unloaded and singly loaded controls. Compilation of 6 independent experiments. (B) Same as panel A but with CD40 staining rather than CD83 (P < .01). Compilation of 4 independent experiments. (C,D) Histograms demonstrating representative results for A (CD83) and B (CD40). (E) Semiquantitative RT-PCR demonstrates differential expression of CTLA-4 between DCs doubly loaded with matched antigens and DCs doubly loaded with mismatched antigens. Matched equals DC loaded with matched class I and II antigens. Mismatched equals DC loaded with mismatched class I and II antigens. +/−, +/− reverse transcriptase.

Figure 4

Aspects of the HA peptide model system. (A) Positioning of MHC class I and II peptides along the primary sequence of influenza HA antigen. (B) Representation of the class I/II overlapping peptide pair (B8-166/DR3-162), the sequence comparison of which is predicted to be disrupted by ethanolamine. The control class I/II overlapping peptide pair (A2-443/DR3-440) is predicted to be unaffected by the presence of ethanolamine.

Figure 5

A system of overlapping/nonoverlapping defined class I and II peptides demonstrates that enhanced Th-1 responses mediated by DC are dependent upon sequence overlap of loaded class I and II antigenic epitopes. (A) Full class I/II sequence overlap. (B) Partial (5 residues) class I/II sequence overlap. (C) Peptides defined as B8- and DR3-restricted are somewhat promiscuous and can elicit responses in an HLA-B8⁻/HLA-DRβ1*03⁻ background (P < .004 in this representative experiment). For panels A, B, and C: equals IFN-γ release induced by DC loaded with overlapping class I and II peptides, □ equals IFN-γ release induced by DC loaded with nonoverlapping class I and II peptides or singly loaded DC; y-axis equals total IFN-γ release in (in μM²). (D) DCs loaded with overlapping class I and II peptide epitopes support enhanced production of activated CD8⁺ cells (P < .001). equals percent CD8⁺CD25⁺ cells induced by DC loaded with overlapping class I and II peptides. □ equals percentage of CD8⁺CD25⁺ cells induced by singly loaded DC. ■ equals average percentage of CD8⁺CD25⁺ cells induced by all populations of singly loaded DC; y-axis equals percentage of CD8⁺CD25⁺ cells.

Figure 6

Gene expression signatures of DCs as a function of loading methodology. (A) Signatures of the top 100 most differentially expressed genes. Duplicate results shown. Unloaded, unloaded DCs; mRNA, DC loaded by mRNA electroporation; lysate, DC loaded by incubation with cell lysates; mismatch, DC doubly loaded with mRNA/lysate preparations derived from disparate cell types; both, DC doubly loaded with mRNA/lysate preparations derived from same cell type. (B) Signatures of all 1750 differentially regulated genes.

Figure 7

Treatment of DC with the glycyl-tRNA synthetase inhibitor ethanolamine inhibits Th-1 responses induced by DC loaded with overlapping class I and II MHC binding peptides only when glycine residues are present in the class I/II sequence overlap region. (A) Addition of ethanolamine reduced CD8⁺ production by more than 70% when DC were loaded with the glycine-containing B8-166/DR3-162 peptide pair. CD8⁺ production induced by the non–glycine-containing peptide pair A2-443/DR3-440 was unaffected; y-axis: percentage of CD3⁺CD8⁺ cells. (B) Addition of ethanolamine reduced CD8⁺CD25⁺ production by more than 80% when DC were loaded with the glycine-containing B8-166/DR3-162 peptide pair. CD8⁺CD25⁺ production induced by the nonglycine-containing peptide pair A2-443/DR3-440 was largely unaffected; y-axis: percentage of CD8⁺CD25⁺ cells. For panels A and B, 1 representative experiment of 3 independent experiments is shown.