G. J. P. Fernando

Vaccine-Induced Th1-Type Responses are Dominant over Th2-Type Responses in the Short Term whereas Pre-existing Th2 Responses are Dominant in the Longer Term

The effect of adjuvant on induction of human papillomavirus type 16 E7 protein‐specific cytotoxic T lymphocytes (CTL) and immunoglobulin G (IgG)2a antibody was studied in C57BL/6 J mice immunized with various adjuvants and E7 protein. Quil‐A adjuvant, but not complete Freunds adjuvant (CFA) or Algammulin, induced a T‐helper 1 (Th1)‐type response to E7, which was characterized by CTL activity against a tumour cell line transfected with E7 protein and by E7‐specific IgG2a. All tested adjuvants elicited comparable levels of E7‐specific IgG1. The longest duration and greatest magnitude of CTL response was seen following two immunizations with the highest dose of E7 and Quil‐A. Simultaneous immunization with a Th1 and a T helper 2 (Th2)‐promoting adjuvant gave a Th1‐type response. However, E7 and Quil‐A were unable to induce a Th1‐type response (as measured by the inability to generate anti‐E7 IgG2a antibody) in animals with a pre‐existing Th2‐type response to E7. These results suggest that saponin adjuvants may be suitable for immunotherapy in humans where a Th1‐type response is sought, provided that there is no pre‐existing Th2‐type response to the antigen.

A model of peripheral CD8(+) T cell tolerance against skin antigen

Induction of immune cell death is one of the many mechanisms used by tumors to evade immune recognition. Here we assessed for the presence of spontaneous apoptosis in blood dendritic cells (DC; LinHLA-DR+ cells) from patients with breast cancer. We document the presence of a significantly (p < 0.05) higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DC in patients with early stage breast cancer (Stage I-II; n ¼ 13) compared to healthy volunteers (n ¼ 15). We examined the role of tumor products on this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DC in PBMC cultures. Aiming to identify factors that protect these cells from apoptosis, we then compared a range of clinically available maturation stimuli including, inflammatory cytokines (TNF- a, IL-1 b, IL-6 and PGE 2; CC); synthetic double stranded RNA (poly I:C) and soluble CD40 ligand. While inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DC from apoptosis. In contrast, CD40 stimulation induced strong up-regulation of the antigen presenting machinery, secretion of IL-12 and protected blood DC through sustained expression of Bcl-2. Exogenous IL-12 also protected blood DC from apoptosis through sustained expression of Bcl-2, suggesting that CD40L-protection could be mediated, at least in part, through IL-12 secretion. Cumulatively our results demonstrate spontaneous apoptosis of blood DC in patients with breast cancer and confirm that ex vivo conditioning of blood DC can protect them from tumor-induced apoptosis.Diverse infectious and inflammatory environmental triggers, through unknown mechanisms, initiate autoimmune disease in genetically predisposed individuals. Here we show that IL-1b, a key cytokine mediator of the inflammatory response, suppresses CD25+CD4+ regulatory T cell function. Surprisingly, suppression by IL-1b occurs only where antigen is presented simultaneously to CD25+CD4+ T cells and to CD25CD4+ antigen-specific effector T cells. Further, NOD mice show an intrinsic over-production of IL-1 that contributes to reduced CD25+CD4+ regulatory T cell function. Thus, inflammation or constitutive over-expression of IL-1b in a genetically predisposed host can initiate a positive feedback loop licensing autoantigen-specific effector cells to inhibit the regulatory T cells maintaining tolerance to self.Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.The delivery of exogenous antigen to antigen presenting cells (APC) for processing and presentation is the first step in the generation of immune responses. We have utilized mannan or mannose as a vehicle to target protein and peptide antigens to mannose binding receptors on antigen presenting cells. In these studies antigen (protein or peptides) conjugated to oxidized mannan (OxMan) generated cellular immune responses in mice whilst antigen conjugated to reduced mannan (RedMan) gave humoral immune responses. These differential immune responses are due to the ability of OxMan to deliver exogenous conjugated antigen to the cytoplasm of APC (macrophages and DC). We have now used OxMan and RedMan to deliver DNA and RNA to APC. Mannan conjugates of poly-lysine (PLL) and polyethyleneimine (PEI) successfully complexed with DNA and RNA as evidenced by retardation on agarose gel electrophoresis. OxMan-PEI or PLL complexed with DNA or RNA transfected mannose receptor expressing J774 cells as well as bone marrow-derived dendritic cells. Mice immunized with OxMan-PLL-DNA conjugate were protected from a challenge of OVA expressing tumour cells. The combination of mannose receptor targeting and immunomodulating properties of OxMan results in an excellent adjuvant/delivery system.Cancer immunotherapy trials conducted over the last few years have concentrated on the analysis of immunological markers of response to antigenically well defined vaccines. Improvements in the quantity or quality of T cell responses, in a patient population, are proposed to allow a rational basis for improved clinical outcomes. However, using current methodologies, only weak immune correlates of clinical response have been found. Further, little attention has been given to other patient or tumour characteristics predisposing to favourable clinical responses following immunotherapy. Establishing such correlates is fundamental to understanding how vaccines work. We have followed this line of investigation with a matured, autologous, dendritic cell/irradiated melanoma cell vaccine for Stage IV melanoma patientsMost of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.