Smith, Shannon E.

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.

Abstract Previous studies have established that 4-1BB and OX40, members of the TNFR family, enhance antitumor immune responses. Recent studies from our laboratory indicate that intratumoral injections of CpG-ODN induce antitumor responses in Her-2/neu tolerant mice. However, this treatment alone is not sufficient to completely eradicate tumors. Here we investigate whether the combination of CpG+anti-OX40 or anti-4-1BB enhance the antitumor responses in neu mice. Our results indicate that CpG+anti-4-1BB but not CpG+anti-OX40 eradicate tumor in neu mice. CpG+anti-4-1BB treatment significantly increased the number of CD8 cells within lymphoid organs as compared to control or anti-OX40 treated animals. More importantly, we observed that anti-4-1BB but not anti-OX40 inhibits the suppressive function of Tregs. Microarray analysis of Tregs revealed specific transcriptional differences brought about by treatment with anti-OX40 or anti-4-1BB. 4-1BB modulates differential gene expression in Tregs of over 300 genes that remain unaffected by OX40. Potential gene candidates include interleukin-9 and protein-tyrosine phosphatase receptor-type zeta-1, which are transcriptionally down and up regulated in the presence of anti-4-1BB. These proteins may play a role in Treg function, and could explain the enhanced anti-tumor effect observed in the presence of anti-4-1BB.

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.