C3d adjuvant effects are mediated through the activation of C3d-specific autoreactive T cells

Abstract

Complement fragment C3d covalently attached to antigens enhances immune responses, particularly for antigens lacking T-cell epitopes. Enhancement has been attributed to receptor cross-linking between complement receptor CR2 (CD21) and polysaccharide antigen to surface IgM on naïve B cells. Paradoxically, C3d has still been shown to increase immune responses in CD21 knockout mice, suggesting that an auxiliary activation pathway exists. In prior studies, we demonstrated the CD21-independent C3d adjuvant effect might be due to T-cell recognition of C3d T-helper epitopes processed and presented by major histocompatibility complex class II on the B-cell surface. C3d peptide sequences containing concentrated clusters of putative human C3 T-cell epitopes were identified using the epitope-mapping algorithm, EpiMatrix. These peptide sequences were synthesized and shown in vitro to bind multiple human leukocyte antigen (HLA)-DR alleles with high affinity, and induce interferon-γ responses in healthy donor peripheral blood mononuclear cells. In the present studies, we establish further correlations between HLA binding and HLA-specific lymphocyte reactions with select epitope clusters. In addition, we show that the T-cell phenotype of C3d-specific reactive T cells is CD4(+)CD45RO(+) memory T cells. Finally, mutation of a single T-cell epitope residing within the P28 peptide segment of C3d resulted in significantly diminished adjuvant activity in BALB/c mice. Collectively, these studies support the hypothesis that the paradoxical enhancement of immune responses by C3d in the absence of CD21 is due to internalization and processing of C3d into peptides that activate autoreactive CD4(+) T-helper cells in the context of HLA class II.

Figure 1. The T cell epitope region of C3d residues 223–246 overlaps with P28

The T cell epitope shared between P28 and C3d_223–246 is shown in bold. Amino acids in italics and underlined were mutated. Solid underline denotes amino acids mutated to alanine in P28Mod1. The additional K>N mutation in P28Mod2 is denoted by the dotted underline. The P28 region, which contains the major binding site of C3d to CD21, shares 13 amino acids with C3d.

Figure 2. PBMCs from healthy donors respond specifically to C3d epitope clusters

Frozen PBMC samples from four healthy donors (A–D) were thawed and stimulated for 10 days with either a pool of human C3d epitope peptides or a non-specific control peptide, PADRE. Cells were re-stimulated in a 48-hour IFNγ ELISpot with either individual peptides or pools. A response was considered positive if the number of spots was greater than or equal to 50 spot forming cells over background per 10⁶ PBMCs. HLA type of donors is shown at the top of each graph. PBMCs from all donors responded to C3d peptides when cultured with the C3d peptide pool.

Figure 2. PBMCs from healthy donors respond specifically to C3d epitope clusters

Frozen PBMC samples from four healthy donors (A–D) were thawed and stimulated for 10 days with either a pool of human C3d epitope peptides or a non-specific control peptide, PADRE. Cells were re-stimulated in a 48-hour IFNγ ELISpot with either individual peptides or pools. A response was considered positive if the number of spots was greater than or equal to 50 spot forming cells over background per 10⁶ PBMCs. HLA type of donors is shown at the top of each graph. PBMCs from all donors responded to C3d peptides when cultured with the C3d peptide pool.

Figure 2. PBMCs from healthy donors respond specifically to C3d epitope clusters

Frozen PBMC samples from four healthy donors (A–D) were thawed and stimulated for 10 days with either a pool of human C3d epitope peptides or a non-specific control peptide, PADRE. Cells were re-stimulated in a 48-hour IFNγ ELISpot with either individual peptides or pools. A response was considered positive if the number of spots was greater than or equal to 50 spot forming cells over background per 10⁶ PBMCs. HLA type of donors is shown at the top of each graph. PBMCs from all donors responded to C3d peptides when cultured with the C3d peptide pool.

Figure 2. PBMCs from healthy donors respond specifically to C3d epitope clusters

Frozen PBMC samples from four healthy donors (A–D) were thawed and stimulated for 10 days with either a pool of human C3d epitope peptides or a non-specific control peptide, PADRE. Cells were re-stimulated in a 48-hour IFNγ ELISpot with either individual peptides or pools. A response was considered positive if the number of spots was greater than or equal to 50 spot forming cells over background per 10⁶ PBMCs. HLA type of donors is shown at the top of each graph. PBMCs from all donors responded to C3d peptides when cultured with the C3d peptide pool.

Figure 3. C3d epitopes specifically induce a memory T cell response

Primary stimulations were performed as in Figure 2. Secondary stimulations with media, C3d peptide pool, MOG_35–55 or CytoStim (Miltenyi) were performed for three hours. IFNγ-secreting cells were then captured, magnetically separated (Miltenyi) and labeled with antibodies for CD4, CD45RA (naïve marker) and CD45RO (memory marker). Cells were first gated for CD4+IFNγ+, then CD45RA and CD45RO. Each primary stimulation group above is subtracted for background with secondary stimulation of media alone. Incubation and re-stimulation with the C3d peptide pool produced a significant increase in the CD4+IFNγ+CD45RO+ memory T cell subpopulation.

Figure 4. Targeted mutation of the single T cell epitope in P28 abolishes predicted binding to HLA and MHC alleles

EpiMatrix cluster reports show the P28, P28Mod1 and P28Mod2 sequences parsed into overlapping 9-mer frames. Each 9-mer is analyzed for predicted recognition by eight common HLA haplotypes and MHC alleles I-A^b, I-A^d and I-E^d. Z-Scores above 1.64 are predicted to bind respective alleles. Epitope/HLA matches are shown in shaded boxes. Modified amino acids in P28Mod1 and P28Mod2 are underlined. The P28Mod1 variant is predicted to disrupt binding to most MHC alleles, and the more aggressive P28Mod2 variant is predicted to fully disrupt binding.

Figure 5. Mutation of the single T cell epitope in P28 significantly diminishes its adjuvant activity

The immunogenicity of wild-type gp120 (wtgp120; open circles) is enhanced when conjugated to a 4X concatamer of P28 (wtgp120-p28; black circles) as measured by anti-Env titers. When conjugated to a 4X concatamer of P28 mutated at the single predicted T cell epitope (wtgp120-p28mod1, wtgp120-p28mod2; open squares and black squares, respectively) the adjuvant activity of P28 is significantly diminished. P28mod1 alone was used as a negative control. Codon optimized gp120 (cogp120), which expresses 7–10 times more gp120 protein than wtgp120 at the same DNA dose, was used as a positive control. Immunization with ENVgp120 glycoprotein conjugated to p28mod1 or p28mod2 significantly diminished antibody responses.