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

Abstract

Background: Immunogenicity 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.

Methods: Biopsies 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.

Results: VSME 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.

Conclusions: These 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).

Keywords: adjuvants, pharmaceutic; arginase; cytokines; immunogenicity, vaccine; melanoma.

Figure 1

Expression of select genes within the vaccination site microenvironment following treatment with different vaccine compositions. Expression data are provided in terms of normalized counts. bars demonstrate median and IQR. n=27. IFA, incomplete Freund’s adjuvant; P, peptide; w, week. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2

Multiparameter immunofluorescence histology of the vaccine site microenvironment postvaccination with peptides in IFA and pICLC showing co-expression of CD40L and CD3 but not CD8. Selected markers include Dapi (blue), CD40L (red), CD3 (yellow), CD8 (green). (A) Includes CD40L and (B) excludes CD40L. White arrows depict CD3⁺CD8^neg T cells co-expressing CD40L and CD3 but not CD8. Red arrows depict cells expressing CD40L but not CD3 or CD8.

Figure 3

Expression of T-cell markers, transcription factors and markers of exhaustion within the vaccination site microenvironment following treatment with different vaccine compositions. Expression data are provided in terms of normalized counts. Bars demonstrate median and IQR. n=27. IFA, incomplete Freund’s adjuvant; P, peptide; w, week. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4

Results of GAGE pathway analysis performed on the complete set of differentially expressed genes under each vaccination condition. Q-values are provided for each enriched pathway under the various vaccination conditions. If a Q-value is not provided, the pathway was not enriched by the associated vaccination condition. IFA, incomplete Freund’s adjuvant; P, peptide; w, week.

Figure 5

Expression of arginase-1 (A), TICAM1 (TRIF) (B) and MYD88 (C) within the vaccination site microenvironment following treatment with different vaccine compositions. Expression data are provided in terms of normalized counts. Bars demonstrate median and IQR. n=27. IFA, incomplete Freund’s adjuvant; P, peptide; w, week. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6

Gene set enrichment analysis highlighting the varied pattern of downregulation of arginine and proline metabolism following single vaccination with peptides in pICLC (A) and repeated vaccination with peptides in IFA (B). ARG1 is not meaningfully downregulated by peptides in pICLC (A) but is strongly downregulated in repeated vaccination with peptides in IFA (B) as shown by its position in the ranked list of genes. Both conditions were compared with normal skin control. Gene set enrichment data were generated using GAGE and included genes from the arginine and proline metabolism pathway. ARG1, arginase-1; IFA, incomplete Freund’s adjuvant; pICLC, poly ICLC; p, peptide.