Gemcitabine depletes regulatory T-cells in human and mice and enhances triggering of vaccine-specific cytotoxic T-cells.

Particle-mediated epidermal delivery (PMED) is a potent genetic vaccination method. However, a recent report found PMED only poorly and infrequently triggered antigen-specific cytotoxic T-cells in cancer patients. Here, we show that injection of the chemotherapeutic drug Gemcitabine in mice results in improvement of the efficacy of subsequent PMED vaccination against NY-ESO-1. We found in mice and in cancer patients that administration of Gemcitabine induces a transient reduction in the percentage of regulatory T-cells among CD4-positive cells. The higher relative sensitivity of regulatory T-cells compared to other CD4-positive T-cells toward cytostatic drugs can be linked to the higher frequency of proliferating cells in the regulatory compartment compared to the nonregulatory CD4-compartment in healthy people and cancer patients. Thus, by affecting regulatory T-cells more than other lymphocyte subsets, chemotherapeutic agents can create a transient hyperimmunoreactive window. Such a window would provide an ideal timepoint to administer a vaccine expected to induce a therapeutically relevant anticancer cytotoxic T-cell response.

The form of NY-ESO-1 antigen has an impact on the clinical efficacy of anti-tumor vaccination.

Anti-tumor vaccination is being evaluated as a prophylactic and therapeutic strategy against cancer growth, dissemination (spreading) or recurrence. Although a large number of studies investigate in detail the identity of antigens to be used for efficacious immune intervention, there have been few studies investigating the optimal form for antigen to be used in the vaccine. Here we show in a mouse H-2(d) MHC background and for NY-ESO-1 that genetic (plasmid DNA) but not full length protein vaccine is capable of inducing a protective prophylactic anti-tumor cytotoxic T-cell immune response in vivo. Peptide vaccination using nominal MHC class I epitope adjuvanted with a Toll Like Receptor agonist such as stabilized RNA can also provide some anti-tumor protection. Our results highlight the idea that when evaluating the clinical efficacy of a cancer vaccine, not only the identity of the antigen but also the format of the vaccine is of the utmost importance.

Particle size and activation threshold: a new dimension of danger signaling.

Previous studies have shown that single-stranded RNA (ssRNA) mixed with protamine forms particles and activates immune cells through Toll-like receptors (TLRs). We have found that the size of protamine-RNA particles generated depends on the electrolyte content when mixing the 2 components. Moreover, we have evidenced that (1) nanometric particles induce production of interferon-alpha, whereas (2) micrometric particles mainly induce production of tumor necrosis factor-alpha (TNF-alpha) in human immune cells. We found that the mechanisms underlying these observations are (1) nanoparticles but not microparticles are selectively phagocytosed by plasmacytoid dendritic cells (pDCs), which produce interferon-alpha and (2) monocytes that produce TNF-alpha have a higher activation threshold than that of pDCs. Thus, at the same time as sensing pathogen-associated molecular patterns such as ssRNA, the immune system distinguishes the size of the associated structure in such a way as to trigger the adapted antivirus (nanometric) or antibacterial/antifungal (micrometric) immune response. Our results introduce a new dimension in danger signaling--how size qualitatively affects innate response.

An essential role for the stalk region of CD8 beta in the coreceptor function of CD8.

The CD8alphabeta heterodimer is integral to the selection of the class I-restricted lineage in the thymus; however, the contribution of the CD8beta chain to coreceptor function is poorly understood. To understand whether the CD8beta membrane proximal stalk region played a role in coreceptor function, we substituted it with the corresponding sequence from the CD8alpha polypeptide and expressed the hybrid molecule in transgenic mice in place of endogenous CD8beta. Although the stalk-swapped CD8beta was expressed on the cell surface as a disulfide-bonded heterodimer at equivalent levels of expression to an endogenous CD8beta molecule, it failed to restore selection of CD8(+) class I MHC-restricted T cells and it altered the response of peripheral T cells. Thus, the stalk region of the CD8beta polypeptide has an essential role in ensuring functionality of the CD8alphabeta heterodimer and its replacement compromises the interaction of CD8 with peptide-MHC complexes.

Antigen kinetics determines immune reactivity.

A current paradigm in immunology is that the strength of T cell responses is governed by antigen dose, localization, and costimulatory signals. This study investigates the influence of antigen kinetics on CD8 T cell responses in mice. A fixed cumulative antigen dose was administered by different schedules to produce distinct dose-kinetics. Antigenic stimulation increasing exponentially over days was a stronger stimulus for CD8 T cells and antiviral immunity than a single dose or multiple dosing with daily equal doses. The same was observed for dendritic cell vaccination, with regard to T cell and anti-tumor responses, and for T cells stimulated in vitro. In conclusion, stimulation kinetics per se was shown to be a separate parameter of immunogenicity. These findings warrant a revision of current immunization models and have implications for vaccine development and immunotherapy.

CD8alpha/alpha homodimers fail to function as co-receptor for a CD8-dependent TCR.

In this study, we have started to dissect the molecular basis of CD8 dependence of a high and low avidity CTL clone specific for the same peptide epitope. Using anti-CD8alpha and anti-CD8beta antibodies, we found that cytotoxicity and IFN-gamma production by high but not by low avidity CTL was strongly CD8 dependent. We isolated the TCR genes of both types of CTL clones and used retroviral gene transfer to analyse the function of these TCR in primary T cells of wild-type and CD8beta-deficient mice. Both TCR triggered antigen-specific killing in wild-type T cells, and blocking experiments showed that CD8 dependence/independence co-transferred with the TCR into primary T cells, indicating that it was dictated by the TCR itself. Gene transfer experiments into CD8beta-deficient T cells revealed that only the TCR derived from the CD8-independent CTL clone elicited antigen-specific cytotoxicity, while the CD8-dependent TCR was non-functional in the absence of the CD8beta-chain. These data indicate a striking difference between CD8alpha/beta heterodimers and CD8alpha/alpha homodimers as only the former were able to provide co-receptor function for the CD8-dependent TCR.

Immunotherapeutic targeting of allergic disease.

The last decades have shown an increasing incidence of allergic illnesses such as rhinoconjunctivitis, with a prevalence of 20-30% in some industrialised parts of the world. The only treatment that may change the natural course of allergic disease is allergen-specific immunotherapy (SIT), which has been shown to prevent the development of asthma in rhinitic patients and anaphylaxis in insect venom allergic patients. However, the risk-benefit ratio for subcutaneous immunotherapy has changed little from when it was first developed in 1911. Novel developments of adjuvants, and allergens as well as methods of administration, now offer improvements in both the efficacy and safety of SIT. This review describes and discusses these new developments in the context of the many recent advances in our understanding of the mechanisms by which immunotherapy appears to act.