Manrique, Soraya Zorro

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 Macrophages with suppressive characteristics such as Tumor associated macrophages or Myeloid derived suppressor cells have been defined showing regulatory function under inflammatory or polarizing conditions. Until now, there are no reports indicating that under normal, non-inflammatory or non-polarizing conditions, macrophages could have immunoregulatory functions. Utilizing knock-in Foxp3-GFP mice, we identified a new population of Foxp3 positive cells that were CD11b+F4/80+ cells. These cells were observed in spleen, lymph nodes, bone marrow and thymus. Similar to Tregs, the CD11b+F4/80+Foxp3+ macrophages inhibited T cell proliferation, whereas Foxp3neg macrophages did not. The Foxp3neg macrophages could be converted to express Foxp3. These induced-CD11b+F4/80+Foxp3+ cells displayed inhibitory properties similar to natural Foxp3pos macrophages. Based on the immunoregulatory properties of these cells, we termed them Macrophage regulatory cells (Mac-regs). These Mac-regs express CD68hiF4/80hiCTLA4hiGITRhiIL-4Rhi. Interestingly, GR-1 is not a differentiation marker. The cytokine, chemokine, growth factor and genomic profiles of Mac-regs are different compared to CD11b+F4/80+Foxp3-cells. For the first time, we demonstrated the occurrence of a natural population of macrophages displaying regulatory function which may contribute to the homeostatic balance of the immune system. This research was supported by Grants from the NIH and AFAR.

Abstract Effective anti-tumor immunotherapy must overcome the immunosuppressive networks created by the tumor. Previous studies from our group indicate that intratumoral injection of CpG-ODN reduces Treg levels within the tumor by 80%. This decrease in Tregs is mainly mediated by the activation of antigen presenting cells secreting IL-6. To explore the effect of IL6 on Tregs, an expression microarray analysis of genes involved in TGFÎ2 dependent Treg conversion, which are also sensitive to IL6, was done. It yielded gene candidates which can be exploited to modulate the function of Tregs. In vitro analysis of two such candidates: chemokine ligand 1 (CCL1) and integrinαE (ITGαE), shows that blocking these proteins reduces de novo conversion of Tregs (CCL1 and ITGαE) and inhibits the suppressive function of Tregs (CCL1 only). In vivo assays using the neu tolerant breast cancer mouse model demonstrates that injection of αCCL1 or αITGαE blocking antibodies significantly reduced the level of Tregs within the tumor and the tumor draining lymph nodes. Furthermore, the combination of intratumoral injections of CpG-ODN and αCCL1 induce complete tumor rejection in neu mice. These results demonstrate that blockage of CCL1 signaling modulated the function of Tregs and subsequently enhances immunotherapeutic approaches.

Regulatory T cells (T reg cells) are characterized by the expression of the forkhead lineage-specific transcription factor Foxp3, and their main function is to suppress T cells. While evaluating T reg cells, we identified a population of Foxp3-positive cells that were CD11b+F4/80+CD68+, indicating macrophage origin. These cells were observed in spleen, lymph nodes, bone marrow, thymus, liver, and other tissues of naive animals. To characterize this subpopulation of macrophages, we devised a strategy to purify CD11b+F4/80+Foxp3+ macrophages using Foxp3-GFP mice. Analysis of CD11b+F4/80+Foxp3+ macrophage function indicated that these cells inhibited the proliferation of T cells, whereas Foxp3− macrophages did not. Suppression of T cell proliferation was mediated through soluble factors. Foxp3− macrophages acquired Foxp3 expression after activation, which conferred inhibitory properties that were indistinguishable from natural Foxp3+ macrophages. The cytokine and transcriptional profiles of Foxp3+ macrophages were distinct from those of Foxp3− macrophages, indicating that these cells have different biological functions. Functional in vivo analyses indicated that CD11b+F4/80+Foxp3+ macrophages are important in tumor promotion and the induction of T reg cell conversion. For the first time, these studies demonstrate the existence of a distinct subpopulation of naturally occurring macrophage regulatory cells in which expression of Foxp3 correlates with suppressive function.

Abstract Intratumoral accumulation of T regulatory cells (Tregs) creates an immunosuppressive environment that reduces the efficacy of antitumor immunotherapy. The immunosuppressive milieu within tumors is largely brought about by the presence of Tregs, which maintain self-tolerance by directly inhibiting T cells, NK cells, and dendritic cells. Depletion of Tregs enhances antitumor immune responses; however, current depletion therapies also affect the function of CD4 and CD8 T effector cells. Previous studies from our laboratory indicate that intratumoral delivery of CpG-ODN strongly reduces the levels of Tregs within the tumor, which is mainly mediated by IL-6. Because IL-6 promotes growth of some human cancers, alternate pathways to inactivate Tregs were sought through microarray analysis, resulting in gene candidates that can be exploited to modulate the function of Tregs. Analysis of these candidates indicates that neutralization of chemokine (C-C motif) ligand 1 (CCL1) prevented de novo conversion and suppressive function of Tregs without affecting the function of T effector cells. The combination of CpG-ODN and anti-CCL1 treatments induced complete rejection of tumors in BALB-neuT tolerant mice, and result in the generation of long-term protective memory responses. Tumor rejection correlated with changes in the lymphocyte composition within the tumor; we observed decreased Treg numbers and a concomitant accumulation of tumoricidal cells such as CD8+NKG2D+ and NK cells. These studies demonstrate that neutralization of CCL1 can be used as an adjuvant to antitumor immunotherapy, as a means of reversing the immunosuppressive function of Tregs without compromising T cell effector function.

Abstract We have shown that neu transgenic mice are immunotolerant and that immunizations with dendritic cells (DC) pulsed with neu-derived antigens were not able to control tumor growth in these animals. We tested whether, by modulating the tumor microenvironment with Toll-like receptor ligands, it could be possible to induce the activation of antitumor responses in neu mice. Our results indicate that only intratumoral (i.t.) injections of CpG-ODN induce an antitumor response in neu mice. To target the CpG-ODN to the tumor site anywhere within the body, we chemically conjugated an anti–Her-2/neu monoclonal antibody (mAb) with CpG-ODN. The anti–neu-CpG hybrid molecule retained its ability to bind to Her-2/neu+ tumors, activate DCs, and induce antitumor responses. Our results indicated that injections of anti–neu-CpG induced the rejection of primary tumors in 100% of BALB/c mice and only in ∼30% of BALB-neuT mice. After challenging the BALB/c and BALB-neuT mice, we observed that BALB/c mice developed a protective memory response; in contrast, BALB-neuT mice succumbed to the challenge. After injections of anti–neu-CpG, T regulatory cells (T-reg) were drastically reduced at the tumor site, but a large number were still present in the lymphoid organs. When BALB-neuT mice were treated with anti–neu-CpG plus anti-GITR mAb, but not with anti-CD25 mAb, 100% of the BALB-neuT mice rejected the primary tumor and developed a protective memory response indicating the critical role of T-regs in regulating the repertoire against self antigens. Taken together, these results indicate that CpG-ODN–targeted therapy and depletion of T-regs optimally activate a primary response and generate a protective memory response against self-tumor antigens. [Cancer Res 2008;68(18):7530–40]