Lustgarten, Joseph

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 T cell responses are compromised in the elderly. The B7-CD28 family receptors are critical in the regulation of immune responses. We evaluated whether the B7-family and CD28-family receptors were differentially expressed in dendritic cells, macrophages, and CD4+ and CD8+ T cells from young and old mice, which could contribute to the immune dysfunction in the old. Although most of the receptors were equally expressed in all cells, >85% of the old naive CD8+ T cells expressed B7-H1 compared with 25% in the young. Considering that B7-H1 negatively regulates immune responses, we hypothesized that expression of B7-H1 would downregulate the function of old CD8+ T cells. Old CD8+ T cells showed reduced ability to proliferate, but blockade of B7-H1 restored the proliferative capacity of old CD8+ T cells to a level similar to young CD8+ T cells. In vivo blockade of B7-H1 restored antitumor responses against the B7-H1− BM-185–enhanced GFP tumor, such that old animals responded with the same efficiency as young mice. Our data also indicate that old CD8+ T cells express lower levels of TCR compared with young CD8+ T cells. However, following antigenic stimulation in the presence of B7-H1 blockade, the levels of TCR expression were restored in old CD8+ T cells, which correlated with stronger T cell activation. These studies demonstrated that expression of B7-H1 in old CD8+ T cells impairs the proper activation of these cells and that blockade of B7-H1 could be critical to optimally stimulate a CD8 T cell response in the old.

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]

AbstractCancer statistics show a disproportionately higher burden of tumors in the old. Most of the studies evaluating vaccination strategies have not taken into consideration the effect that aging has on the immune system. For the first time, we describe an animal tumor model in which self-tolerance and aging are present at the same time. FVB-Her-2/neu mice which are tolerant to neu antigens crossed with HLA-A2/Kb mice (A2xneu) develop spontaneous tumors when they are more than 22 months old. Analysis of CD8+ T-cell–specific responses in A2xneu mice indicated that the priming activity of old A2xneu mice to induce an immune response was diminished compared with young animals. Following intratumoral injections of CpG-ODN, ∼30% of young A2xneu mice rejected the tumor; however, no antitumor effect was observed in old A2xneu mice. Analysis of T regulatory cells (Treg) indicated that there are significantly more Tregs in old animals. After CpG-ODN vaccination plus Treg depletion, 70% of young A2xneu mice rejected the tumor. The same treatment prolonged survival in old A2xneu mice, but none of the animals rejected the tumor. Even though CpG-ODN injections plus Treg depletion could rescue the antitumor responses against self-tumor antigens in young tolerant mice, the same therapy is not as effective in old tolerant hosts. Relevant tumor models such as the A2xneu mice in which self-tolerance and aging are present at the same time are critical to allow the optimization of vaccination strategies to effectively stimulate immune responses against self-tumor antigens in the young and the old. [Cancer Res 2008;68(13):5423–31]