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In previous studies we have shown that Signal Transducer and Activator of Transcription 3 (STAT3) negatively regulates inflammatory responses in myeloid cells and plays a central role in determining immune activation versus immune tolerance of antigen-specific CD4+ T-cells. Indeed, in Stat3 knock out mice (LysMcre/Stat3flox/−) in which macrophages and neutrophils are devoid of Stat3, we found that in response to a tolerogenic stimulus (high dose peptide-induced tolerance or tumor-induced tolerance models) adoptively transferred antigen-specific CD4+ T-cells are not tolerized but instead are effectively primed as determined by their production of IL-2 and IFN-gamma in response to cognate antigen. Such an observation led us to investigate which cell population(s) is required for the priming effect observed in Stat3 KO mice. First, we used anti-Gr-1 antibody to deplete neutrophils in wild type BALB/c mice as well as Stat3 KO mice. Briefly, half the mice in each group were treated with 0.5mg of the antibody given i.p. every 3 days from day-3 until day +15. On day zero, all the mice were adoptively transferred with 2.5 x 106 naïve transgenic CD4+ T-cells specific for a MHC class II restricted epitope of hemaglutinin (HA). On day +2, animals received high dose of HA peptide (275 mcg) given i.v. Mice were sacrificed on day +15 and clonotypic T-cells were re-isolated from their spleens to assess their functional status following their in vivo exposure to this tolerogenic stimuli. A striking difference was observed in T-cells isolated from Stat3 KO mice with an intact neutrophil compartment (non-depleted) versus T-cells from anti-Gr-1 treated LysMcreStat3flox/− mice. Unlike T-cells from the former group in which priming was the functional outcome, clonotypic T-cells from LysMcreStat3flox/− mice depleted of neutrophils, were found to be anergic. Therefore, the T-cell priming effect observed in LysMcreStat3flox/− mice requires an intact neutrophil compartment given that in the absence of this population, tolerance not priming was the functional T-cell outcome. To gain insight into the potential mechanism(s) by which neutrophils devoid of Stat3 influence T-cell responses, we next analyzed the phenotypic and functional properties of neutrophils isolated from Stat3 KO mice and wild type controls. First, the lack of expression of MHC class II molecules by neutrophils from WT and KO mice made unlikely the possibility that neutrophils devoid of Stat3 could directly present antigen to CD4+ T-cells. However, when neutrophils from Stat3−/− conditional mice were added to macrophages monolayers in vitro, the antigen-presenting capabilities of macrophages was significantly enhanced as determined by the increased production of IL-2 and IFN-gamma by antigen-specific T-cells encountering cognate antigen in these APCs. Furthermore, macrophages cultured in vitro with neutrophils from Stat3−/− conditional mice were able to restore the responsiveness of tolerant CD4+ T-cells. This effect that was not observed when tolerized T-cells encountered cognate antigen in macrophages incubated with neutrophils from wild type mice. Trans-well experiments demonstrated that the regulatory effect of neutrophils upon APCs function required cell-cell contact. Taken together, we have unveiled a previously unrecognized role of neutrophils in determining the functional outcome of antigen-specific T-cell responses, effect that is dependent upon the interaction of neutrophils with antigen-presenting cells.

A large number of CD4+ T-cells from either aged mice or humans display surface markers associated with an activated/memory phenotype. In spite of these changes however, these T-cells have a markedly decreased ability to proliferate and produce IL-2 in response to antigen stimulation in vitro. The cellular and molecular mechanisms involved in this age-related unresponsiveness of the CD4+ T-cell compartment remain poorly understood. Utilizing a well-established experimental system in which transgenic CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA) are adoptively transferred into non-transgenic recipients, we have previously elucidated important mechanisms involved in the induction and maintenance of CD4+ T-cell tolerance. Our studies were however limited to the analysis of T-cell function in lymphoma bearing young mice (4 to 10 weeks old). Here, we assessed the influence of the aged microenvironment in determining the phenotype and function of antigen-specific T-cells. CD4+ T-cells from young TCR transgenic mice (2 months old) were adoptively transferred into either old (20–24 months) or young (2 months old) non-transgenic mice. Two weeks later, clonotypic and non-clonotypic CD4+ T-cells were isolated from the spleens of these animals and their phenotype and function were determined in vitro. Reminiscent of the age-related changes observed within the normal CD4+ T-cell repertoire, young transgenic T-cells transferred into aged hosts have acquired an activated/memory phenotype but displayed a significant impairment in antigen-specific proliferation and IL-2 production in response to cognate antigen in vitro. These changes were not due to homeostatic proliferation of the transferred T-cells into the relatively lymphopenic aged host. To determine whether the changes observed in “aged” T-cells were reversible or not, we adoptively transfer old T-cells back into young hosts or into control old mice. While old transgenic T-cells transferred into an old environment remained fully unresponsive, the adoptive transfer of the same old T-cells into a young host restored their ability to proliferate and produce IL-2. Surprisingly, these “old” T-cells were able to produce significantly higher levels of IFN-gamma indicative of their memory/effector phenotype. Furthermore, young animals adoptively transferred with “aged” antigen-specific T-cells were now capable of rejecting A20 B-cell lymphomas expressing HA as a model tumor antigen (A20HA). Taking together, factor(s) present in the aged microenvironment are responsible for limiting the effector function of CD4+ T-cells that seem otherwise well equipped to become fully activated if the proper environment is provided (young microenvironment). The potential role of soluble suppressive factors as well as regulatory T-cells (Tregs) in the unresponsiveness observed in the T-cell compartment of aged hosts will be discussed.

There is now overwhelming evidence that tumor-induced antigen (Ag)-specific T cell tolerance represents a critical problem in tumor immunology. Early studies of CD8 T cell tolerance equated peripheral tolerance with either ignorance or clonal deletion, although more recent evidence has suggested that this may be only partly accurate. While murine modeling outwardly supports the contention that high-affinity tumor-specific CD8 T cell responses are centrally deleted, cognate CD8 T cells displaying an Ag-experienced phenotype can nonetheless be detected in regional draining lymph nodes (dLN) or in non-lymphoid sites where the Ag is present. However, these CD8 T cells are typically deficient in one or more effector functions, including cytokine production, cytotoxicity, or proliferative capacity. To better define the state of Ag-specific CD8 T cell responsiveness in the face of progressive tumor, we adoptively transferred hemagglutinin (HA) Ag-specific Clone 4 (CLN4) CD8 T cells into animals bearing a genetically modified B cell lymphoma expressing HA as a model tumor antigen (A20HA). Analysis of the fate and function of these transferred antigen-specific CD8 T cells revealed that they encountered antigen in vivo, were capable of mounting an initial response to A20HA but this response was not sustained. Indeed, while a prominent CTL activation was observed in the spleen and draining lymph nodes of tumor bearing mice within 14 days of T cell transfer, responses (HA-specific proliferation, IFN-γ production and cytotoxicity) began to wane by day 21 after T cell transfer, and in particular their ability to produce IFN-γ. A similar pattern of transient activation followed by loss of CD8 T cell function has been also observed in an in vivo model of high-dose peptide induced antigen-specific CD8 T cell tolerance. Given our recent demonstration that the disruption of Stat3 signaling in APCs overcomes CD4 T cell tolerance we determined next whether Stat3 deficient APCs may be inherently better at cross-presenting tumor-Ags and elicit therefore a more productive and sustained CD8 T cell response. In an in vitro system in which tumor cells expressing a model tumor antigen (EL4mOVA) were cultured with APCs genetically devoid of Stat3 signaling and anti-OVA CD8 T-cells (OT-I), we found that these T cells displayed an enhanced function relative to antigen-specific CD8 T-cells that encountered antigen on APCs with an intact Stat3 signaling. Currently, we are investigating whether CD8 T-cell tolerance to tumor antigens occurred -or not- in tumor bearing mice with a genetic disruption of Stat3 signaling in APCs. Furthermore, given the emerging role of other members of the STAT family in regulation of APC function, we are exploring whether targeted disruption of Stat1, 4 and 6 can alter the ability of the CD8 T-cell to sustain a protective response or, more importantly to recover function once tolerance is induced.

The immune system is very effective in fighting infections but seems not to be as efficient in recognizing and destroying cancer cells. In the cancer setting, tumor antigen uptake and presentation by APCs to antigen-specific T-cells often occurs in the absence of inflammation resulting therefore in tolerance induction. It is plausible therefore that by converting APCs from a non-inflammatory to an inflammatory phenotype through ligation of TLRs we may well overcome immune tolerance and tip the balance towards productive tumor antigen-specific T-cell responses. Among all the TLR-ligands identified to date, flagellin is the only one with a strictly proteinic nature, characteristic that make it a suitable candidate for cloning and transfection into tumor cells to generate novel tumor cell based vaccines. In this study, we first evaluated whether treatment with purified flagellin could prevent tolerance induction in vivo. Naïve CD4+ T-cells (2.5x106) specific for a MHC class II-restricted epitope of influenza hemagglutinin (HA) were adoptively transferred intravenously into BALB/c mice, 24 hours after mice were given either a tolerogenic dose of HA-peptide (200 mg), or a combination of this high dose of peptide together with Flagellin (10 mg I.V.). Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses towards the cognate antigen evaluated in vitro. As expected clonotypic T-cells isolated from animals treated with high dose peptide were fully tolerant, in sharp contrast with those isolated from flagellin treated animals that displayed normal responses in terms of cytokine production and proliferation. Surprisingly, this preservation of T-cell function following in vivo treatment with flagellin was not observed when animals were treated with high dose HA-peptide in the presence of the TLR4 ligand, LPS. To better understand the mechanism(s) by which flagellin, and not LPS, preserved the responsiveness of antigen-specific CD4+ T-cells to cognate antigen presented by APCs, we assessed the phenotypic characteristics and the cytokine profile of macrophages and DCs treated in vitro with these TLRs ligands. Although LPS-treated APCs produce higher levels of IL-12, relative to flagellin-treated APCs, the production of this pro-inflammatory cytokine was accompanied by a parallel induction of the anti-inflammatory cytokine, IL-10. Interestingly, flagellin-treated APCs produced IL-12 but were unable to produce IL-10. This effect was dependent on ligation of TLR5, since it was not observed when RAW264.7 cells -which lack TLR5- were treated with flagellin. In vivo studies further confirmed our observations since IL-10 was not detected in the serum of animals treated with flagellin, but it was present in significant amounts in LPS-treated animals. This inhibitory effect of flagellin on IL-10 production was seen even when APCs were stimulated in vitro with strong inducers of IL-10. Given the above properties of flagellin, we generated two novel approaches to be used in the formulation of tumor cell based vaccines: 1) A bystander cell line transfected with the fliC gene from Salmonella typhimurim for flagellin expression (B78H1-Flagellin) and 2) Microspheres beads coated with flagellin. Both vaccination strategies are being currently studied in the in vivo and in vitro settings