Incomplete Freund’s adjuvant reduces arginase and enhances Th1 dominance, TLR signaling and CD40 ligand expression in the vaccine site microenvironment

Abstract

BackgroundImmunogenicity of cancer vaccines is impacted by adjuvants and schedule, but systematic assessments of their effects have not been performed. Montanide ISA-51, an incomplete Freund’s adjuvant (IFA), is used in many vaccine trials, but concerns have been raised about negative effects in murine studies. We found in humans that IFA enhances systemic immune responses and that repeat vaccination at one site (same site vaccination (SSV)) creates tertiary lymphoid structures (TLS) in the vaccine site microenvironment (VSME). We hypothesized that vaccination with peptides+IFA+pICLC or SSV×3 with peptides in IFA would create an immunogenic milieu locally at the VSME, with activated dendritic cells (DC), TLS-associated chemokines and a Th1-dominant VSME.MethodsBiopsies of the VSME were obtained from participants on two clinical trials who were immunized with multiple melanoma peptides (MELITAC 12.1) in adjuvants comprising IFA and/or the TLR3-agonist pICLC. Biopsies were obtained either a week after one vaccine or a week after SSV×3. Controls included normal skin and skin injected with IFA without peptides. Gene expression analysis was performed by RNAseq.ResultsVSME samples were evaluated from 27 patients. One vaccine with peptides in pICLC+IFA enhanced expression of CD80, CD83, CD86 (p<0.01), CD40 and CD40L (p<0.0001) over normal skin; these effects were significantly enhanced for SSV with peptides+IFA. Vaccines containing pICLC increased expression of TBX21 (T-bet) but did not decrease GATA3 over normal skin, whereas SSV with peptides in IFA dramatically enhanced TBX21 and decreased GATA3, with high expression of IFNγ and STAT1. SSV with peptides in IFA also reduced arginase-1 (ARG1) expression and enhanced expression of TLR adapter molecules TICAM-1 (TRIF) and MYD88. Furthermore, SSV with IFA and peptides also enhanced expression of chemokines associated with TLS formation.ConclusionsThese findings suggest that SSV with peptides in IFA enhances CD40L expression by CD4 T cells, supports a Th1 microenvironment, with accumulation of activated and mature DC. Increased expression of TLR adaptor proteins after SSV with peptides in IFA might implicate effects of the skin microbiome. Reduced ARG1 may reflect diminished suppressive myeloid activity in the VSME.Trial registration number(NCT00705640,NCT01585350).

Insights into tertiary lymphoid structures in the solid tumor microenvironment: anti-tumor mechanism, functional regulation, and immunotherapeutic strategies

“…Another clinical trial evaluated the antitumor effects of vaccination with multiple melanoma peptides in adjuvants comprising incomplete freund’s adjuvant (IFA) and/or the TLR3-agonist pICLC. After repeated vaccination with peptides in IFA, the vaccine site microenvironment showed dramatically enhanced T-bet and decreased GATA3, with high expressions of IFNγ and STAT1, and reduced arginase-1 expression and enhanced expressions of chemokines associated with TLS formation 78 . An intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens also induced a robust tissue-localized effector immune response in 12 CIN2/3 patients 79 .…”

HLA-A2.1-restricted ECM1-derived epitope LA through DC cross-activation priming CD8+ T and NK cells: a novel therapeutic tumour vaccine

“…Besides, peptide mixed with adjuvants is a rational option to compensate for the immune response deficiency of peptide application alone [48]. ISA is an ideal immune stimulatory adjuvant [49]. We prepared formulations of ISA-51 [50] and LA or YL, which could enhance the stability of epitopes in vivo, to immunize HLA-A2.1 transgenic mice for the collection of splenic cells to further inoculate into tumour-bearing NOD/SCID mice for immunological reconstitution.…”

Fast Maturation of Splenic Dendritic Cells Upon TBI Is Associated With FLT3/FLT3L Signaling

“…Therefore, with the demonstration of increased FLT3 phosphorylation, upregulation of protein synthesis markers, and increased expression of MHC-II and lysosomal proteins LAMP1 and CD68, we believe to provide substantial evidence to state that TBI induces a quick upregulation of the splenic DC maturation process, from steady-state cells to effective APCs. Several other markers are commonly applied to the study of DC maturation, such as CD40, CD80, and CD86 (97), which are often upregulated along with MHC-II [e.g., (98)(99)(100)]. These markers were not included in the present study and further characterization of the phenotype of splenic DC cells activated upon TBI may reveal their dynamics.…”