Cohen, Peter A.

Abstract Chronic inflammation in the aged closely resembles tumor-induced immune suppression, manifested as a progressive T1→T2 shift and an increased presence of MDSCs. We hypothesize that reversing these age-related phenomena should reduce the elderly’s heightened susceptibility to malignancy. We have now optimized real-time analyses of phosphorylated STATs in murine T cells and MDSCs in order to correlate STAT activation with immune suppression. Both young and old T cells responded to IL-4 stimulation with STAT6 activation and to IFN-γ stimulation with STAT1 activation. Uniquely, however, young T cells also phosphorylated STAT1, STAT3 and STAT5 in response to IL-4, and responded to IL-6 with STAT3 activation, all consistent with a greater susceptibility to T1/T17 differentiation. Regarding MDSCs, both old and young displayed constitutive STAT3 activation which could be enhanced by G-CSF or IL-6 treatment. Both young and old MDSCs activated STAT1 briskly in response to IFN-γ, STAT5 in response to GM-CSF and STAT6 in response to IL-4. However, younger MDSCs, like younger T cells, displayed more diverse responsiveness to IL-4, activating not only STAT6 but also STAT1 and STAT3. While chronic in vivo exposure to T2 cytokines may explain the decreased responsiveness of older MDSCs and T cells to IL-4, paradoxically IL-4 still robustly activated STAT6 in these cells. Such selective responsiveness may be required to preserve the immune suppressive potential of older MDSCs.

Abstract IL-9 is a cytokine that is primarily associated with mast cells and airway inflammation, however it also plays a role in T regulatory cell (Treg) survival and recruitment to tumors. We previously reported that 4-1BB treatment of Tregs inhibits both Treg function and IL-9 secretion. Neutralization of IL-9 with an anti-IL-9 antibody inhibits the suppressive function of Tregs without affecting the function of CD4+ and CD8+ T effector cells. Furthermore, the combination of intra-tumoral CpG and anti-IL-9 induced tumor rejection in BALB-neuT and MUC1 tolerant transgenic mice. Here we show that the lack of IL-9 inhibits Treg suppression of T cell proliferation, using IL-9 knockout (IL-9ko) mice. Phenotypic analysis of Treg markers showed that tumor draining lymph nodes of 4T1 bearing mice contain fewer CTLA-4+ and CD103+ Tregs, and that these also have reduced of CTLA-4 and CD103 expression. We also observed that IL-9ko mice spontaneously reject TUBO and 4T1 mammary carcinoma cells and that rejection is abrogated by CD8+ T cell depletion. Finally, preliminary studies of human CD4+ T cell proliferation revealed that IL-9 neutralization led to greater proliferation than isotype control treated cells. Taken together our results suggest that IL-9 can have an immunosuppressive function that comes into play in the tumor microenvironment, and that the blockade of IL-9 could serve as a novel strategy to perturb the function of Tregs to enhance the antitumor effect of tumor vaccines.

Abstract Although tumors from melanoma patients can often provide a source of therapeutically active T cells for adoptive therapy, it has historically proved challenging to employ peripheral blood as the T cell source for any type of cancer. We have observed nonetheless that HER2-specific T cells can be Ag-driven and enriched in culture when PBMCs from HER2-vaccinated breast cancer patients are treated with exogenous IL12. We hypothesized that stimulating innate immunity could further improve PBMC cultures by activating the DC subpopulation more effectively than adding exogenous rIL12. Bulk PBMC exposure to optimized TLR agonists produced vast quantities of IL12 and IL23, and upregulated HLA-DR, B7.1 and CD40 in the DC subpopulation, effects not produced by exogenous IL12. Subsequent IL7 exposure emulated homeostatic proliferation, selectively causing the Ag-driven T cell subset both to proliferate faster and to strongly resist apoptosis. Ag-specificity within two weeks approached 80-100% of both CD4+ and CD8+ T cells for recall Ags and 1-15% for a wide array of MUC1- and HER2-derived peptides, including the ability to distinguish glycoforms. Culture-expanded T cells retained a young CD28+/CD56- phenotype, uniformly expressed ROR-γ during culture, variably produced IL-2 and/or IL-17, and uniformly expressed T-bet and secreted IFN-γ when reexposed to Ag. Such T1/T17 bipotency may be ideal for anti-tumor adoptive therapy and highly efficient for use in Ag discovery.

Abstract Stimulation of dendritic cells (DCs) by exogenous danger signals such as single Toll-like receptor (TLR) ligands results in generalized DC activation. Recently, Napolitani and co-workers showed select TLR ligand pairings induced a more specialized DC activation program including enhanced IL-12 p70 secretion and the capacity to polarize CD4 T cells into IFN-γ-secreting Th1 cells. We therefore examined the effect of pairing an exogenous TLR2 ligand (lipoteichoic acid) with an endogenous danger signal, ATP, which is found in high extracellular concentrations in damaged tissues, malignant tumors, and possibly other pathophysiological settings. ATP added to LTA modified somewhat the expression of common DC activation markers, but primarily induced a distinct pattern of cytokines with greatly enhanced levels of both IL-23 and IL-1β, while TNF, IL-10 and IL-6 remained mostly unchanged. IL-12 was not produced. These DC induced a Th17 response when stimulating allogeneic T cells in a manner partially dependent on IL-1β. These studies show that pairing of endogenous and exogenous danger signals can induce a specialized DC activation program that may have important physiological implications.

Abstract Chronic inflammation associated with aging is termed inflamm-aging. It manifests as a rise in many proinflammatory mediators and contributes to a decline in immune function. Unregulated inflammasome activation initiates an inappropriate inflammatory response. It is particularly imperative to understand inflammasome dysregulation in the elderly (>65 yrs), since it is a likely basis of immune dysfunction. Analyses of freshly isolated memory CD4+ and CD8+ splenic T cells demonstrated that even without further activation, inflammasome genes were already significantly up-regulated in old mice compared to young, including factors critical to myeloid-derived suppressor cell (MDSC) expansion including S100a8, S100a9, IL-1β, IL-13 and IL-6. A corresponding 2-3 fold increase in CD11b+Gr1+cells was observed in the spleens of old naïve mice compared to young. Importantly, cells with the CD11b+Gr1+ phenotype from old mice, unlike young, were functionally MDSCs and suppressed T cell proliferation. Therefore, the immunosuppressive environment of the tumor-bearing host also appears to be operative in aging, even in the absence of tumor. We hypothesize that constituitive activation of inflammasome complex results in a chronic inflammatory state, altering immune responses in the elderly similarly to the tumor-bearing state. Preemptive reversal of inflammasome-induced MDSCs may enhance T cell immunosurveillance in the elderly, a population with a heightened susceptibility to malignancy.

AbstractA fully synthetic MUC1‐based cancer vaccine was designed and chemically synthesized containing an endogenous helper T‐epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody‐dependent cell‐mediated cytotoxicity (ADCC). It also activated cytotoxic T‐lymphocytes. Collectively, the immunological data demonstrate engagement of helper T‐cells in immune activation. A synthetic methodology was developed for a penta‐glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1‐based cancer vaccines that elicited potent immune responses employed exogenous helper T‐epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T‐epitope will be more attractive, because it will activate cognate CD4+ T‐cells that will provide critical tumor‐specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.