Marcel Camps

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

Defects in major histocompatibility complex (MHC) class I–restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.

Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity

Dendritic cells (DCs) are crucial for priming of naive CD8+ T lymphocytes to exogenous antigens, so-called “cross-priming.” We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I–peptide complexes at the cell surface.

Abrogation of CTL epitope processing by single amino acid substitution flanking the C-terminal proteasome cleavage site.

CTL directed against the Moloney murine leukemia virus (MuLV) epitope SSWDFITV recognize Moloney MuLV-induced tumor cells, but do not recognize cells transformed by the closely related Friend MuLV. The potential Friend MuLV epitope has strong sequence homology with Moloney MuLV and only differs in one amino acid within the CTL epitope and one amino acid just outside the epitope. We now show that failure to recognize Friend MuLV-transformed tumor cells is based on a defect in proteasome-mediated processing of the Friend epitope which is due to a single amino acid substitution (N→D) immediately flanking the C-terminal anchor residue of the epitope. Proteasome-mediated digestion analysis of a synthetic 26-mer peptide derived from the Friend sequence shows that cleavage takes place predominantly C-terminal of D, instead of V as is the case for the Moloney MuLV sequence. Therefore, the C terminus of the epitope is not properly generated. Epitope-containing peptide fragments extended with an additional C-terminal D are not efficiently translocated by TAP and do not show significant binding affinity to MHC class I-Kb molecules. Thus, a potential CTL epitope present in the Friend virus sequence is not properly processed and presented because of a natural flanking aspartic acid that obliterates the correct C-terminal cleavage site. This constitutes a novel way to subvert proteasome-mediated generation of proper antigenic peptide fragments.

Immune Complex-Loaded Dendritic Cells Are Superior to Soluble Immune Complexes as Antitumor Vaccine

Dendritic cells (DCs) play an important role in the induction of T cell responses. FcγRs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual FcγRs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that FcγRI and FcγRIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating FcγRs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating FcγRs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.

The Inhibiting Fc Receptor for IgG, FcγRIIB, Is a Modifier of Autoimmune Susceptibility

FcγRIIB-deficient mice generated in 129 background (FcγRIIB129−/−) if back-crossed into C57BL/6 background exhibit a hyperactive phenotype and develop lethal lupus. Both in mice and humans, the Fcγr2b gene is located within a genomic interval on chromosome 1 associated with lupus susceptibility. In mice, the 129-derived haplotype of this interval, named Sle16, causes loss of self-tolerance in the context of the B6 genome, hampering the analysis of the specific contribution of FcγRIIB deficiency to the development of lupus in FcγRIIB129−/− mice. Moreover, in humans genetic linkage studies revealed contradictory results regarding the association of “loss of function” mutations in the Fcγr2b gene and susceptibility to systemic lupus erythematosis. In this study, we demonstrate that FcγRIIB−/− mice generated by gene targeting in B6-derived ES cells (FcγRIIBB6−/−), lacking the 129-derived flanking Sle16 region, exhibit a hyperactive phenotype but fail to develop lupus indicating that in FcγRIIB129−/− mice, not FcγRIIB deficiency but epistatic interactions between the C57BL/6 genome and the 129-derived Fcγr2b flanking region cause loss of tolerance. The contribution to the development of autoimmune disease by the resulting autoreactive B cells is amplified by the absence of FcγRIIB, culminating in lethal lupus. In the presence of the Yaa lupus-susceptibility locus, FcγRIIBB6−/− mice do develop lethal lupus, confirming that FcγRIIB deficiency only amplifies spontaneous autoimmunity determined by other loci.

Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T‐cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre‐)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte‐derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4+ and CD8+ T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasome‐ and transporter associated with Ag processing‐dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8+ T‐cell activation.

Differential expression regulation of the alpha and beta subunits of the PA28 proteasome activator in mature dendritic cells.

Activation of dendritic cells (DC) by Th-dependent (CD40) or -independent (LPS, CpG, or immune complexes) agonistic stimuli strongly enhances the expression of the proteasome activator PA28αβ complex. Upon activation of DC, increased MHC class I presentation occurred of the melanocyte-associated epitope tyrosinase-related protein 2180-188 in a PA28αβ-dependent manner. In contrast to other cell types, regulation of PA28αβ expression in DC after maturation was found to be IFN-γ independent. In the present study, we show that expression of PA28α and β subunits was differentially regulated. Firstly, PA28α expression is high in both immature and mature DC. In contrast, PA28β expression is low in immature DC and strongly increased in mature DC. Secondly, we show the presence of a functional NF-κB site in the PA28β promoter, which is absent in the PA28α promoter, indicating regulation of PA28β expression by transcription factors of the NF-κB family. In addition, glycerol gradient analysis of DC lysates revealed elevated PA28αβ complex formation upon maturation. Thus, induction of PA28β expression allows proper PA28αβ complex formation, thereby enhancing proteasome activity in activated DC. Therefore, maturation of DC not only improves costimulation but also MHC class I processing. This mechanism enhances the CD8+ CTL (cross)-priming capacity of mature DC.

Efficient Induction of Antitumor Immunity by Synthetic Toll-like Receptor Ligand–Peptide Conjugates

Zom, Khan, Britten, and colleagues report that direct conjugation of lipopeptide Pam3CSK4 to synthetic long peptides enhanced in vivo targeting and maturation of the conjugate with superior priming of CD8+ and CD4+ T cells in two mouse tumor models. Chemical conjugates comprising synthetic Toll-like receptor ligands (TLR-L) covalently bound to antigenic synthetic long peptides (SLP) are attractive vaccine modalities, which can induce robust CD8+ T-cell immune responses. Previously, we have shown that the mechanism underlying the power of TLR-L SLP conjugates is improved delivery of the antigen together with a dendritic cell activation signal. In the present study, we have expanded the approach to tumor-specific CD4+ as well as CD8+ T-cell responses and in vivo studies in two nonrelated aggressive tumor models. We show that TLR2-L SLP conjugates have superior mouse CD8+ and CD4+ T-cell priming capacity compared with free SLPs injected together with a free TLR2-L. Vaccination with TLR2-L SLP conjugates leads to efficient induction of antitumor immunity in mice challenged with aggressive transplantable melanoma or lymphoma. Our data indicate that TLR2-L SLP conjugates are suitable to promote integrated antigen-specific CD8+ and CD4+ T-cell responses required for the antitumor effects. Collectively, these data show that TLR2-L SLP conjugates are promising synthetic vaccine candidates for active immunotherapy against cancer. Cancer Immunol Res; 2(8); 756–64. ©2014 AACR.

Dendritic cells, but not macrophages or B cells, activate major histocompatibility complex class II-restricted CD4 + T cells upon immune-complex uptake in vivo

Professional antigen‐presenting cells (APC) are able to process and present exogenous antigen leading to the activation of T cells. Antigen–immunoglobulin (Ig)G complexes (IC) are much more efficiently processed and presented than soluble antigen. Dendritic cells (DC) are known for their ability to take up and process immune complex (IC) via FcγR, and they have been shown to play a crucial role in IC‐processing onto major histocompatibility complex (MHC) class I as they contain a specialized cross‐presenting transport system required for MHC class I antigen‐processing. However, the MHC class II‐antigen‐processing pathway is distinct. Therefore various other professional APC, like macrophages and B cells, all displaying FcγR, are thought to present IC‐delivered antigen in MHC class II. Nonetheless, the relative contribution of these APC in IC‐facilitated antigen‐presentation for MHC class II in vivo is not known. Here we show that, in mice, both macrophages and DC, but not B cells, efficiently capture IC. However, only DC, but not macrophages, efficiently activate antigen‐specific MHC class II restricted CD4+ T cells. These results indicate that mainly DC and not other professional APC, despite expressing FcγR and MHC class II, contribute significantly to IC‐facilitated T cell activation in vivo under steady‐state conditions.

Fcγ Receptor IIb Strongly Regulates Fcγ Receptor-Facilitated T Cell Activation by Dendritic Cells

FcγR ligation by Ag–Ab immune complexes (IC) not only mediates effective Ag uptake, but also strongly initiates dendritic cell (DC) maturation, a requirement for effective T cell activation. Besides the activating FcγRI, FcγRIII, and FcγRIV, the inhibitory FcγRIIb is expressed on DCs. It is unclear how the ratio between signals from the activating FcγR and the inhibitory FcγRIIb determines the outcome of FcγR ligation on DCs. By microarray analysis, we compared the transcriptomes of steady state and IC-activated bone marrow-derived wild-type (WT) DCs expressing all FcγR or DCs expressing only activating FcγR (FcγRIIb knockout [KO]) or only the inhibitory FcγRIIb (FcR γ-chain KO). In WT DCs, we observed a gene expression profile associated with effective T cell activation, which was absent in FcR γ-chain KO, but strikingly more pronounced in FcγRIIb KO bone marrow-derived DCs. These microarray results, confirmed at the protein level for many cytokines and other immunological relevant genes, demonstrate that the transcriptome of IC-activated DCs is dependent on the presence of the activating FcγR and that the modulation of the expression of the majority of the genes was strongly regulated by FcγRIIb. Our data suggest that FcγRIIb-deficient DCs have an improved capacity to activate naive T lymphocytes. This was confirmed by their enhanced FcγR-dependent Ag presentation and in vivo induction of CD8+ T cell expansion compared with WT DCs. Our findings underscore the potency of FcγR ligation on DCs for the effective induction of T cell immunity by ICs and the strong regulatory role of FcγRIIb.