Joseph Lustgarten

Fat tissue, aging, and cellular senescence.

Fat tissue, frequently the largest organ in humans, is at the nexus of mechanisms involved in longevity and age‐related metabolic dysfunction. Fat distribution and function change dramatically throughout life. Obesity is associated with accelerated onset of diseases common in old age, while fat ablation and certain mutations affecting fat increase life span. Fat cells turn over throughout the life span. Fat cell progenitors, preadipocytes, are abundant, closely related to macrophages, and dysdifferentiate in old age, switching into a pro‐inflammatory, tissue‐remodeling, senescent‐like state. Other mesenchymal progenitors also can acquire a pro‐inflammatory, adipocyte‐like phenotype with aging. We propose a hypothetical model in which cellular stress and preadipocyte overutilization with aging induce cellular senescence, leading to impaired adipogenesis, failure to sequester lipotoxic fatty acids, inflammatory cytokine and chemokine generation, and innate and adaptive immune response activation. These pro‐inflammatory processes may amplify each other and have systemic consequences. This model is consistent with recent concepts about cellular senescence as a stress‐responsive, adaptive phenotype that develops through multiple stages, including major metabolic and secretory readjustments, which can spread from cell to cell and can occur at any point during life. Senescence could be an alternative cell fate that develops in response to injury or metabolic dysfunction and might occur in nondividing as well as dividing cells. Consistent with this, a senescent‐like state can develop in preadipocytes and fat cells from young obese individuals. Senescent, pro‐inflammatory cells in fat could have profound clinical consequences because of the large size of the fat organ and its central metabolic role.

Flagellin Fusion Proteins as Adjuvants or Vaccines Induce Specific Immune Responses

ABSTRACT Vaccination is the most efficient prophylaxis against a variety of infectious diseases. New vaccination strategies rely on the incorporation of effective adjuvants, which stimulate the innate immune response and, in turn, activate the adaptive immune response. It is well established that flagellin induces inflammatory responses through the activation of antigen-presenting cells (APCs). In order to evaluate whether flagellin can serve as a carrier for the development of adjuvants or vaccines, we prepared a flagellin-enhanced green fluorescent protein (EGFP) fusion protein. Our results demonstrate that a flagellin-EGFP fusion protein is capable of stimulating APCs, resulting in the maturation of these cells and secretion of proinflammatory cytokines. Furthermore, APCs pulsed with the flagellin-EGFP fusion protein effectively process and present EGFP antigens. More importantly, animals immunized with the flagellin-EGFP fusion protein developed specific anti-EGFP T-cell responses. In contrast, recombinant EGFP was not able to stimulate APCs, nor did it induce a T-cell response. Thus, recombinant-flagellin fusion proteins may be suitable carriers as adjuvants or vaccines for the development of new vaccination strategies to induce and boost immune responses against infectious diseases and cancer.

High Accumulation of T Regulatory Cells Prevents the Activation of Immune Responses in Aged Animals

In our previous in vivo study we demonstrated that young BALB/c mice effectively rejected the BM-185 tumor cells expressing enhanced GFP (EGFP) as a surrogate tumor Ag. In contrast, old BALB/c mice succumbed to the BM-185-EGFP tumors, indicating that there is a deficiency in old animals preventing the rejection of immunogenic tumors. There is cumulative evidence indicating that regulatory T (Treg) cells control the activation of primary and memory T cell responses. However, very little is known about whether there is a relation between Tregs and the lack of immune responses in the aged. We evaluated young and aged animals, and our results demonstrated that there are significantly more CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ Tregs in the spleen and lymph nodes of old animals when compared with the young. Depletion of CD25+ cells with anti-CD25 mAb induces the rejection of BM-185-EGFP cells, restores antitumor T cell cytotoxic activity, and results in the generation of a protective memory response against the BM-185 wild-type tumors in old mice. Furthermore, vaccination with CpG-oligodeoxynucleotide decreases the number of Treg cells in old animals to the same levels as young mice, restoring the primary and memory antitumor immune responses against BM-185-EGFP tumors. Taken together, these results indicate that there is a direct correlation between the expansion of Treg cells and immune deficiency in the old, and that depletion of these cells might be critical for restoring immune responses in aged animals.

Vaccination with dendritic cells pulsed with apoptotic tumors in combination with anti-OX40 and anti-4-1BB monoclonal antibodies induces T cell-mediated protective immunity in Her-2/neu transgenic mice.

Tumor cells express tumor‐associated antigens (TAAs), which can serve as targets for the immune system. However, the majority of TAAs are overexpressed products of normal cellular genes; as such, self‐tolerance mechanisms have hindered their use for the induction of effective antitumor responses. One such normal self‐protein is the growth factor receptor Her‐2/neu, which is overexpressed in 25–35% of all mammary carcinomas in humans. In previous studies, we have demonstrated that Her‐2/neu mice are functionally tolerant to neu antigens and contain only a low avidity T‐cell repertoire to neu antigens. However, this residual low‐avidity T‐cell repertoire has antitumor activity. In this study, we compared the immune responses of Her‐2/neu mice immunized with dendritic cells (DCs) pulsed with soluble neu protein or with apoptotic tumor cells. Analysis of the antitumor response shows that Her‐2/neu mice vaccinated with DCs pulsed with Her‐2/neu antigens retard tumor growth; however, vaccination with DCs pulsed with apoptotic tumor cells induces a stronger antitumor effect. Administration of multiple immunizations in combination with the costimulatory agonist anti‐OX40 or anti‐4‐1BB MAb significantly enhanced the immune responses in these mice, resulting in complete tumor rejection if the tumor burden was small and substantial tumor reduction with a larger tumor burden. These results have important implications for the design of tumor vaccination strategies, suggesting that the use of vaccines that stimulate a broad immune response in combination with costimulatory molecules as immunomodulators could significantly improve the antitumor immune response in tolerant hosts.

The CD8+ T cell repertoire against Her-2/neu antigens in neu transgenic mice is of low avidity with antitumor activity.

The majority of tumor‐associated antigens are aberrantly expressed or overexpressed normal gene products. Therefore, mechanisms responsible for self tolerance dampen immune responses against these antigens. To evaluate the effect that tolerance has on the immune responses against tumor antigens, we characterized the CD8+ Tu2004cell responses in neu mice. Tu2004cell responses against theA2.1/neu p369–377 and p773–782 peptides were evaluated in neu mice that were crossed with A2.1/Kb transgenic mice (A2×neu). Tetramer binding and cytotoxic activity demonstrate that, compared to CTL from A2.1/Kb×FVB wild‐type mice (A2×FVB), CD8+ Tu2004cells from A2×neu mice were of lower avidity for the peptides. Despite the fact that A2×neu mice are tolerant, multipleimmunizations with DC pulsed with the p369–377 or p773–782 peptides in the presence of IL‐2 retarded tumor growth in A2×neu mice, and immunizations in combination with the anti‐OX40 mAb further enhanced the antitumor response. Taken together, these data indicate that low‐avidity Tu2004cells for neu antigens persisting in A2×neu mice have the capacity to develop antitumor responses as long as they are provided with efficient costimulation. These results underscore the potential role of low‐avidity Tu2004cells in antitumor immunity and may offer an important component for vaccination immunotherapies.

Aged mice develop protective antitumor immune responses with appropriate costimulation.

There is a clear decrease in CD8+ T cell effector function with aging, a loss once thought to be intrinsic to the CD8+ T cells. Recent studies suggest, however, that this decline may be a consequence of altered stimulatory signals within the aged lymphoid microenvironment. In this study, we compared the immune responses of young and old mice against the BM-185 pre-B cell lymphoma expressing enhanced GFP (EGFP) as a surrogate tumor Ag. Young animals develop protective immune responses when immunized with BM-185-EGFP, but aged mice do not and ultimately succumb to the tumor. However, expression of CD80 (B7.1) on the BM-185-EGFP (BM-185-EGFP-CD80) results in rejection of the tumor by both young and old animals. Additionally, injection of BM-185-EGFP-CD80 cells in young mice promotes the development of long-lasting memory responses capable of rejecting BM-185 wild-type tumors. Aged animals similarly injected did not develop antitumor memory responses. Interestingly, old animals immunized with the BM-185-EGFP-CD80 cells plus injections of the agonist anti-OX40 mAb did develop long-lasting memory responses capable of rejecting the BM-185 wild-type tumors with the same vigor as the young animals. We show that old mice have the capacity to develop strong antitumor responses and protective memory responses as long as they are provided with efficient costimulation. These results have important implications for the development of vaccination strategies in the elderly, indicating that the aged T cell repertoire can be exploited for the induction of tumor immunity.

Aging affect the anti-tumor potential of dendritic cell vaccination, but it can be overcome by co-stimulation with anti-OX40 or anti-4-1BB.

It has been well established that there is a decline in immune function with age resulting in a diminished capacity to respond to infections or tumors. Although many studies have demonstrated the efficacy of autologous dendritic cells (DC) vaccines in stimulating an anti-tumor immune response in the young, almost none of these reports consider the effect that aging has on the immune system or test whether DC-vaccination is effective in old hosts. In this study we compared the efficacy of DC-vaccination in young and old mice. Our results showed that DC-vaccination in young animals induced an anti-tumor response resulting in approximately 60% tumor growth inhibition, while minimal protection was observed in old animals. DC vaccination plus rIL-2 further enhanced the anti-tumor response in young animals (approximately 70-75% inhibition), while ineffective in old animals. In contrast, co-administration of anti-OX-40 or anti-4-1BB mAbs vigorously enhanced the anti-tumor immune response in both young (approximately 85-90% inhibition) and old mice (approximately 70-75% inhibition). Our data indicate that although old mice have a decline in immune function, they have the capacity to develop strong anti-tumor responses as long as they are provided with efficient co-stimulation.

Foxp3-positive macrophages display immunosuppressive properties and promote tumor growth

Identification of a population of Foxp3-expressing suppressive macrophages.

Maximal T Cell–Mediated Antitumor Responses Rely upon CCR5 Expression in Both CD4+ and CD8+ T Cells

Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.

Blockade of CCL1 Inhibits T Regulatory Cell Suppressive Function Enhancing Tumor Immunity without Affecting T Effector Responses

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.