Anti-TBX21 Monoclonal antibody, clone 5C20

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 SSVx3 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 SSVx3. 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 IFN gamma 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.

In order to analyze the effect of vitamin E on Th1 and Th2 cytokine production, porcine peripheral blood mononuclear cells (PBMC) were isolated from healthy pigs (n = 8) and cultured with either 0, 10, 50, or 100 mu M of vitamin E (alpha-tocopherol). PBMC were stimulated with PHA for either, 24 h to determine: (a) the concentration of tocopherol incorporated into the cell membrane, (b) cytokine production and (c) Th1 and Th2 regulators gene expression; or 72 h to determine the proliferation of PBMC. Vitamin E was incorporated into the PBMC in a dose dependent manner, giving as a result a high proliferation of cells irrespective of the dose of vitamin E used. Regarding cytokine production, vitamin E consistently decreases the mRNA expression and the percentage of cells producing IL-10. Vitamin E did not influence the production of IFN-gamma but the lowest level of vitamin E (10 mu M) was sufficient to maximally increase the proportion of cells producing IL-2, to diminish IL-4, and discreetly increase the mRNA expression of TBX21 vs. GATA3. In conclusion, our results revealed that vitamin E is able to suppress IL-10 production and to influence the production of IL-2, IL-4, and maybe TBX21. Vitamin E clearly has immunomodulatory effects, though further work in vivo to determine the physiological nature of these effects is warranted. (c) 2009 Elsevier B.V. All rights reserved.