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Supplementary Figure 2 from Systemic Targeting of CpG-ODN to the Tumor Microenvironment with Anti–neu-CpG Hybrid Molecule and T Regulatory Cell Depletion Induces Memory Responses in BALB-neuT Tolerant Mice

Supplementary Figures 1-10 from Maximal T Cell–Mediated Antitumor Responses Rely upon CCR5 Expression in Both CD4+ and CD8+ T Cells

Supplementary Figure 3 from Systemic Targeting of CpG-ODN to the Tumor Microenvironment with Anti–neu-CpG Hybrid Molecule and T Regulatory Cell Depletion Induces Memory Responses in BALB-neuT Tolerant Mice

Supplementary Figure 1 from Systemic Targeting of CpG-ODN to the Tumor Microenvironment with Anti–neu-CpG Hybrid Molecule and T Regulatory Cell Depletion Induces Memory Responses in BALB-neuT Tolerant Mice

PurposeHigh calorie intake leads to obesity, a global socio-economic and health problem, reaching epidemic proportion in children and adolescents. Saturated and monounsaturated fatty acids from animal (lard) fat are major components of the western-pattern diet and its regular consumption leads to obesity, a risk factor for cardiovascular disease. However, no clear evidence exists whether consumption of diet rich in saturated (SFAs) and monounsaturated (MUFAs) fatty acids has detrimental effects on cardiac structure and energetics primarily due to excessive calories. We, therefore, sought to determine the impact of high calories versus fat content in diet on cardiac structure and mitochondrial energetics.MethodsSix-week-old C57BL/6J mice were fed with high calorie, high lard fat-based diet (60% fat, HFD), high-calorie and low lard fat-based diet (10% fat, LFD), and lower-calorie and fat diet (standard chow, 12% fat, SCD) for 10 weeks.ResultsThe HFD- and LFD-fed mice had higher body weight, ventricular mass and thickness of posterior and septal wall with increased cardiomyocytes diameter compared to the SCD-fed mice. These changes were associated with a reduction in the mitochondrial oxidative phosphorylation (OXPHOS) complexes I and III activity compared to the SCD-fed mice without significant differences between the HFD- and LFD-fed animals. The HFD-fed animals had higher level of malondialdehyde (MDA) than LFD and SCD-fed mice.ConclusionsWe assume that changes in cardiac morphology and selective reduction of the OXPHOS complexes activity observed in the HFD- and LFD-fed mice might be related to excessive calories with additional effect of fat content on oxidative stress.

It is well established that immune responses are diminished in the old. However, we still do not have a clear understanding of what dictates the dysfunction of old T cells at the molecular level. Although microarray analysis has been used to compare young and old T cells, identifying hundreds of genes that are differentially expressed among these populations, it has been difficult to utilize this information to pinpoint which biological pathways truly affect the function of aged T cells. To better define differences between young and old naive CD4+ and CD8+ T cells, microarray analysis was performed pre- and post-TCR stimulation for 4, 12, 24 and 72 h. Our data indicate that many genes are differentially expressed in the old compared to the young at all five time points. These genes encode proteins involved in multiple cellular functions such as cell growth, cell cycle, cell death, inflammatory response, cell trafficking, etc. Additionally, the information from this microarray analysis allowed us to underline both intrinsic deficiencies and defects in signaling only seen after activation, such as pathways involving T-cell signaling, cytokine production, and Th2 differentiation in old T cells. With the knowledge gained, we can proceed to design strategies to restore the function of old T cells. Therefore, this microarray analysis approach is a powerful and sensitive tool that reveals the extensive changes seen between young and old CD4+ and CD8+ naive T cells. Evaluation of these differences provides in-depth insight into potential functional and phenotypical differences among these populations.

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.

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.

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.

Clinical and preclinical data indicate that immunotherapeutic interventions could induce immune responses capable of controlling or retard the tumor growth. However, immunotherapies need to be further optimized. We hypothesized that a more effective strategy for tumor eradication is to directly target the tumor microenvironment in order to generate a proinflammatory response and induce a localized antitumor immune response capable of eliminating the tumor cells. Nanoparticles have been proven to be an effective delivery system. In these studies we evaluated conjugated anti-RNEU and anti-CD40 antibodies onto PLA-(poly dl-lactic acid)-biodegradable nanoparticles (PLA-NP) for the induction of antitumor immune responses. The anti-neu/anti-CD40-NP were functional in vitro recognizing RNEU(+) tumors and activating dendritic cells. The delivery of anti-neu/anti-CD40-NP but not anti-neu-NP or anti-CD40-NP induced an antitumor response resulting in complete tumor elimination and generation of protective memory responses. The anti-neu/anti-CD40-NP specifically activated an antitumor response against RNEU(+) tumors but not against RNEU(-) tumors. The antitumor immune responses correlate with the induction of a Th1-proinflammatory response, reduction in the number of Tregs within the tumor and activation of a specific cytotoxic response. These results indicate that anti-neu/anti-CD40-NP with immunomodulatory properties are safe and can be used effectively as cancer vaccines strategy for the specific induction of antitumor immune responses.

Cancer 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]

The deterioration of immune function with advancing age contributes to the increased incidence of morbidity and mortality among the elderly from infectious disease and possibly cancer. The innate and adaptive arms of immunity are affected by the deleterious effects of aging, but it is adaptive immunity, and in particular T lymphocytes, that is most susceptible to these effects. The aging of the immune system, referred to as immunosenescence, is associated with a dramatic decline in responsiveness as well as functional dysregulation. Age-associated alterations in T cells are evident at all stages of its development, and it is these changes that contribute to the overall increased susceptibility to infection and possibly cancer. Although there is an enormous effort worldwide to develop vaccines, much of the research is performed with young animals. Because the immune system of the aged is different from that of the young, many of the findings cannot be extrapolated. In this review, we will discuss those differences in T cell immunity of the aged, relate how these differences influence the immune response of the aged to pathogens as well as to tumors, and describe the current approaches to rejuvenate the aged immune response. Although the majority of the conclusions on T cell immunity in the aged are based on studies in the murine model, many of these findings can be extended to the human model. Throughout this review, we have noted those findings that are specific to humans.

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.

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.

IL-2 is a T-cell growth factor that has pleiotropic functions on T cells, assisting in activating and expanding immune responses. Interestingly, we have previously found that in the presence of IL-2, CD4(+) or CD8(+) T cells kill tumor cells in an antigen-independent and non-MHC-restricted manner. In this study we took advantage of IL-2 capabilities to induce in this way the killing of tumor cells for immunotherpeutic purposes. We describe a novel mechanism where it is possible to bypass the usage of tumor-specific T cells for adoptive transfer by using non-tumor-specific T cells in combination with an anti-Her-2/neu-IL-2 (neu-Ab-IL-2) or heregulin-IL-2 fusion protein for tumor eradication. T cells in the presence of fusion proteins are capable of inducing the lysis of tumor cells in a non-MHC-restricted manner. This lysis is only observed in T cells that have been stimulated through the TCR, and it is mediated through the Fas-FasL pathway. Moreover, transfer of non-tumor-specific T cells in combination with the fusion proteins, induced the eradication of established Her-2, Her-3, and Her-4 expressing tumor cells in SCID mice. In contrast, the combination of non-tumor-specific T cells plus rIL-2 or irrelevant Ab-IL-2 (anti-hemagglutinin-IL-2) does not induce the elimination of tumors, indicating that the antitumor activity is dictated by the specificity of the fusion protein. These data demonstrate that neu-Ab-IL-2 or heregulin-IL-2 fusion proteins can direct the non-tumor-specific T cells to the tumor site and induce the elimination of tumors. Therefore, combination of non-tumor-specific T cells and antibody/ligand-IL-2 fusion proteins provides an alternative therapeutic strategy to control tumor growth in vivo.

Elevated levels of the p53 protein occur in ∼50% of human malignancies, which makes it an excellent target for a broad-spectrum T cell immunotherapy of cancer. A major barrier to the design of p53-specific immunotherapeutics and vaccines, however, is the possibility that T cells may be tolerant of antigens derived from wild-type p53 due to its low level of expression in normal thymus and lymphohemopoetic cells. The combination of p53 deficient (p53−/−) and p53+/+ HLA-A2.1/Kb transgenic mice was used as a model to explore the possibility that A2.1restricted cytotoxic T lymphocytes (CTL) are functionally tolerant of self peptides derived from the wild-type p53 tumor suppressor protein. A2.1-restricted CTL specific for a naturally processed p53 self-epitope spanning residues 187-197 were completely aborted in p53+/+ as opposed to p53−/− transgenic mice. In contrast, CTL specific for a second self-epitope spanning residues 261-269 of the murine p53 sequence were detected in both p53−/− and p53+/+ A2.1/Kb transgenic mice. However, the avidity of the CTL effectors obtained from p53+/+ mice was 10-fold lower than that obtained from p53−/− mice, again suggesting elimination of CTL with high avidity for the A2.1-peptide complex. The circumvention of functional tolerance of high avidity CTL may therefore be a necessary prerequisite for optimizing immunotherapy against A2.1-restricted wild-type p53 epitopes in humans.

The synthesis of IgE antibodies by B cells is the first in a series of steps resulting in an allergic response. To eliminate IgE-bearing B cells and thereby prevent IgE production, we have developed an immunotoxin (ITA) composed of the non-anaphylactic 84.1c anti-mouse IgE mAb and the A chain of ricin (ricin A). This ITA specifically inhibited the induction of IgE synthesis by lipopolysaccharide plus interleukin-4 (LPS + IL-4) in vitro, and antigen-specific IgE production in vivo in adult mice. A single dose of anti-IgE ITA, given within a week (either before or after) of antigen challenge completely abolished antigen-specific primary IgE responses. No IgE production was seen for 2 months after ITA treatment. Following antigenic re-challenge, a suppressed secondary response (over 50% reduction) was still seen in the ITA-treated mice, 100 days after immunization. The results of this study demonstrate the potential use of anti-IgE toxin conjugates for the suppression of periodic (seasonal) allergic outbreaks.

Chimeric receptors containing antibody-derived Fv or scFv as their extracellular recognition elements can redirect the specificity of T cells in an MHC-independent manner. Upon encountering their target cells, such T-bodies are able to undergo specific stimulation for interleukin/cytokine production, and kill hapten-modified or tumor cells in both in vitro and in vivo model systems. T cells expressing chimeric receptors are able to discriminate between antigen-expressing and normal cells, with negligible bystander cytotoxicity. Unlike antibodies, T cells are well suited to penetrate and destroy solid tumors. Further in vivo studies should be carried out to evaluate and optimize the persistence, homing patterns, and reactivation potential of T-bodies in vivo. Several technical obstacles must be overcome before this approach may be applied clinically. A most urgent problem is the low efficiency of T cell transfection techniques and the particular difficulty of transducing primary T cell populations. While retroviral-mediated gene transfer is more efficient than conventional techniques such as electroporation, the proportion of transfected cells remains low, necessitating an enrichment step. In addition, antibodies with improved discrimination between cell-bound and soluble forms of tumor antigens must be obtained to expand the repertoire of tumor antigens which may be targeted. For each antigen-antibody system, the optimal design of the scFv must be determined. In the future application of this technology, the recognition element used for chimeric TCR is not limited to antibody-derived fragments [27]. Various ligands may be coupled to a T cell-triggering molecule in an attempt to redirect cytotoxic function towards target cells expressing a particular receptor molecule. Although still experimental, we feel that with fine tuning, the T-body approach shows promise as an efficient and broad-spectrum modality for tumor immunotherapy.

The variable regions of the T-cell receptor for antigen (TCR) and antibodies serve as recognition elements, enabling T and B cells to bind antigen with high specificity. Nevertheless, the complex recognition requirements of T cells, which respond to antigen only in the context of a self MHC molecule, do not permit the generation of antigen-specific lymphocytes, which could serve as universal effector cells. To enable the generation of non-MHC-restricted T cells of predetermined specificity, we took advantage of the similarity between the antibody and the T-cell molecule and expressed in T-cell lines chimeric T-cell receptor (cTCR) genes in which the TCR variable region is replaced by an antibody variable region of defined specificity. In another design, the antibody recognition unit has been used in the form of a single-chain Fv (scFv) to create hybrid molecules with other receptor subunits, including the ζ chain of the CD3 complex and the γ chain of the high-affinity receptor for IgE (FcϵRI). T-cell lines transfected with the chimeric TCR or scFvR exhibit cytotoxicity or secrete cytokines in response to antigen displayed either on a solid substrate or on the surface of target cells. We describe here the various configurations of the chimeric receptors that we have produced. Technical aspects, including the choice of vector, antibody sequences, and triggering molecule, are reviewed.[ep [rs

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

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. Cancer Res; 71(16); 5455–66. ©2011 AACR.

Identification of a population of Foxp3-expressing suppressive macrophages., 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.

Previous studies from our laboratory indicate that intratumoral (i.t.) injections of CpG-ODN are the most effective adjuvant strategy to induce an antitumor immune response in tolerant BALB-neuT mice but insufficient for tumor eradication. We evaluated whether this treatment strategy could be enhanced by the presence of anti-OX40 and anti-4-1BB antibodies. Treatment with anti-4-1BB resulted in a greater antitumor response than anti-OX40. The results indicate that anti-4-1BB but not anti-OX40 inhibited the suppressive function of T regulatory cells (Tregs). Through microarray analysis we evaluated the mechanism by which anti-4-1BB inhibits iTregs using the Foxp3-GFP mice. We observed specific transcriptional differences in over 100 genes in iTregs treated with anti-4-1BB, and selected those genes that remained unaffected by exposure to anti-OX40. Interleukin 9 was transcriptionally down-regulated 28-fold by anti-4-1BB treatment, and this was matched by a significant reduction of IL-9 secretion by iTregs. Furthermore, blockade of the common γ-chain receptor resulted in the inhibition of iTreg-suppressive function. More importantly, neutralization of IL-9 plus i.t. injections of CpG-ODN induces tumor rejection in BALB-neuT and MUC-1 tolerant transgenic mice. These results indicate that IL-9 plays a role in iTreg biology during the tumor inflammatory process enhancing/promoting the suppressive function of these cells and that the blockade of IL-9 could serve as a novel strategy to modulate the function of Tregs to enhance the antitumor effect of tumor vaccines.

The impact of aging on T-cell tolerance has yet to be elucidated. More importantly cancer vaccines that will be effective both in the young and the old have yet to be developed. As a result, there is a need for relevant tumor models which include aspects of self-tolerance and development of spontaneous tumors in the aged. Such models are critical for the development and optimization of specific cancerrelated immunotherapeutic strategies for the elderly. Although the majority of studies augmenting immune responses against a self-antigen like the Her-2/neu have used young Her-2/neu transgenic mouse models and put a lot of effort into aspects such as increasing the immunogenicity, very little attention has been paid to the immune competence of the aging population. Based on the Her-2/neu transgenic mice, our group has developed a mouse model (HLA-A2.1/Kb mice crossed with the FVBHer- 2/neu mice) where self-tolerance, spontaneous tumor progression and aging are present simultaneously. The immunological aspects of the A2xneu mice closely reflect those of cancer patients whose immune systems are not fully competent to reject their tumors. Models like this are critical as they may provide data that closely predict the clinical outcomes and will help to customize immunotherapeutic strategies that would be effective for the treatment of tumors in both the young and the old. In this chapter, we will focus on the Her-2/neu transgenic mouse model for the evaluation of immune and antitumor responses against a self-tumor antigen in both the young and the old.

Targeted cancer immune therapy , Targeted cancer immune therapy , کتابخانه دیجیتال جندی شاپور اهواز

A very unique and important feature of TLR (toll-like receptor) agonists is their ability to modulate TLR on the cells of the innate immune system resulting in changes that will induce effective and efficient adaptive immune responses. Therefore, stimulation of TLR pathways through their respective ligands presents a potentially attractive approach to activate adaptive anticancer immune responses. In this chapter, we summarize the effect of targeting some of the TLR for the induction of antitumor responses. Although the use of TLR agonists as future vaccine adjuvants seems very promising, we also need to proceed with caution and long term studies will be critical in assessing the propensity of chronic therapy to trigger autoimmune conditions or promote tumor growth.

Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates, in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. We used the rAAV system to induce specific CTLs against tumor antigens for the development of ovarian cancer (OC) gene therapy. As an extension of the versatility of the rAAV system, we incorporated a self-antigen, Her-2/neu, which is expressed in many cancers, including breast and ovarian. We analyzed two different vectors containing a short (157-612) and long domain (1-1197). Our rAAV vector induced strong stimulation of CTLs directed against the self tumor antigen, Her-2/neu. We then investigated the efficiency of the CTLs in killing Her-2/neu-targeted cells. A significant MHC class I-restricted, anti-Her-2/neu-specific CTL killing was demonstrated against Her-2/neu-positive OC cells after one in vitro stimulation. In summary, single peripheral blood mononuclear cell (PBMC) stimulation with rAAV/157-612- or rAAV/1-1197-pulsed DCs induces strong antigen-specific CTL generation. The CTLs were capable of lysing low doses of peptides pulsed into target cells or OC Her-2/neu(+) tumors. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against OC antigens.

Aging of the immune system is associated with a dramatic reduction in responsiveness as well as functional dysregulation. This deterioration of immune function with advancing age is associated with an increased incidence of cancer. Although there is a plethora of reports evaluating the effect of immunotherapy in stimulating antitumor immune responses, the majority of these studies do not pay attention to the effect aging has on the immune system. Studies from our group and others indicate that immunotherapies could be effective in the young, are not necessarily effective in the old. To optimally stimulate an antitumor immune response in the old, it is necessary to (1) identify and understand the intrinsic defects of the old immune system and (2) use relevant models that closely reflect those of cancer patients, where self-tolerance and aging are present simultaneously. The present review summarizes some defects found in the old immune system affecting the activation of antitumor immune responses, the strategies used to activate stronger antitumor immune response in the old and the use of a tolerant animal tumor model to target a self-tumor antigen for the optimization of immunotherapeutic interventions in the old.

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]

There is accumulative evidence indicating that targeting antigen presenting cells (APCs) with different types of adjuvants could result in the induction of antitumor immune responses. It has been hypothesized that APCs function may be altered in the elderly contributing to a decline in the immune function. We evaluated whether targeting APCs following injection with Poly I:C, LPS, flagellin, imiquimod and CpG-ODN would induce an antitumor response in the old.The immune and antitumor responses induce Poly I:C, LPS, flagellin, imiquimod and CpG-ODN were compared in young (2 month old) and old (18 months) mice.Our results indicated that only intratumoral (i.t.) injections of CpG-ODN completely rejected the tumor in both young and old mice. Injections of Poly I:C also induced the rejection of tumors in the young but not in the old. Furthermore, i.t. injections of CpG-ODN promoted the development of protective memory responses in the young and the old. Analysis of the immune responses in the old indicated that CpG-ODN but not Poly-I:C induces: a pro-inflammatory Th1 type response; accumulation and activation of CD4+, CD8+ T and, NK cell responses; activation of APCs; and reduction in the number of Tregs. The activation of these immune-parameters positively correlates with the induction of an antitumor response.These studies indicate that there are differences in the level of stimulation with TLR-ligands between young and old APCs and that the aged immune responses can be rescued and exploited for the induction of tumor immunity by targeting APCs with specific TLR-ligands. These results have important clinical implications for developing immunization strategies containing TLR-ligands that will be effective in both the young and old.

Recognition of tumor-associated Ags (TAAs) on tumor cells by CTLs and the subsequent tumor cell death are assumed to be dependent on TAA protein expression and to correlate directly with the level of peptide displayed in the binding site of the HLA class I molecule. In this study we evaluated whether the levels of Her-2/neu protein expression on human tumor cell lines directly correlate with HLA-A*0201/Her2/neu peptide presentation and CTL recognition. We developed a TCR mimic (TCRm) mAb designated 1B8 that specifically recognizes the HLA-A2.1/Her2/neu peptide (369–377) (Her2(369)-A2) complex. TCRm mAb staining intensity varied for the five human tumor cell lines analyzed, suggesting quantitative differences in levels of the Her2(369)-A2 complex on these cells. Analysis of tumor cell lines pretreated with IFN-γ and TNF-α for Her2/neu protein and HLA-A2 molecule expression did not reveal a direct correlation between the levels of Her2/neu Ag, HLA-A2 molecule, and Her2(369)-A2 complex expression. However, compared with untreated cells, cytokine-treated cell lines showed an increase in Her2(369)-A2 epitope density that directly correlated with enhanced tumor cell death (p = 0.05). Although a trend was observed between tumor cell lysis and the level of the Her2(369)-A2 complex for untreated cells, the association was not significant. These findings suggest that tumor cell susceptibility to CTL-mediated lysis may be predicted based on the level of specific peptide-MHC class I expression rather than on the total level of TAA expression. Further, these studies demonstrate the potential of the TCRm mAb for validation of endogenous HLA-peptide epitopes on tumor cells.

The majority of the currently defined tumor-associated Ags are often overexpressed products of normal cellular genes. Therefore, tolerance deletes high-affinity T cells directed against the TAAs, leaving only a low-affinity repertoire. We have demonstrated previously that the T cell repertoire against the immunodominant p773–782 A2.1-Her-2/neu-restricted peptide has low affinity in A2xneu mice (Her-2/neu mice crossed with A2.1/Kb mice), compared with A2xFVB mice (A2.1/Kb crossed with FVB-wild-type mice). Immunizations with this peptide have a minor impact in preventing tumor growth in A2xneu mice. Therefore, attempts to expand these responses may be of little clinical value. We hypothesized that if not all possible cross-reactive peptides (CPs) are naturally processed and presented, the possibility exists that T cells against these CPs persist in the repertoire and can be used to induce antitumor responses with higher avidity against native epitopes present on the tumor cells. We have used the positional scanning synthetic peptide combinatorial library methodology to screen the p773–782 T cell clone. The screening data identified potential amino acids that can be substituted in the primary sequences of the p773–782 peptide. The designed CPs induce CTL responses of higher affinity in A2xneu mice compared with the native p773–783 peptide. These CTLs recognize A2+-Her-2/neu+ tumors with high efficiency. Moreover, multiple immunizations with CPs significantly prolonged the survival of tumor-bearing A2xneu mice. These results have demonstrated that it was possible to circumvent tolerance with the identification of CPs and that these peptides could be of significant clinical value.

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.

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.

We describe a novel syngeneic "pseudo-orthotopic" in vivo model of prostate cancer progression. Our model uses the dorsal skinfold chamber technique with fluorescence video microscopy and TRAMP-C2 tumor cells. The cells were transfected with a histone H2B-GFP fusion protein, permitting real-time measurement of tumor size, as well as mitotic and apoptotic indices. To generate a "pseudo-orthotopic" milieu, pieces of prostate tissue (10-15 mm2) from donor mice were implanted into the chambers of C57BL/6 mice. The prostate tissue grafted into the chambers retained its native vasculature, as determined by transplantation of prostate tissue from GFP transgenic mice. TRAMP-C2 prostate cancer tumor spheroids (25,000 cells) were implanted in the chamber. Without prostate tissue, TRAMP-C2 prostate tumors were poorly angiogenic, displayed low mitotic and apoptotic indices (0.7 x 10(-4)), and no significant tumor growth could be detected. TRAMP-C2 tumors growing on transplanted prostate tissue in the chamber on the other hand had mitotic indices in the order of 1.6 x 10(-4) and apoptotic indices in the order of 0.8 x 10(-4). Furthermore, tumors with stroma were highly angiogenic, and were fully vascularized within 7-10 days. During a 4-week observation period, the number of tumor cells increased by nearly 300%. We used the model to study the effects of surgical castration. The most profound response was a rapid vascular regression of the tumor vasculature. Castration also increased apoptotic indices within the tumor without significant changes in mitosis. This model may be utilized for the rapid analysis of new therapeutic candidates against prostate cancer.

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+ T cell responses in neu mice. T cell responses against the A2.1/neu p369-377 and p773-782 peptides were evaluated in neu mice that were crossed with A2.1/Kb transgenic mice (A2 x neu). Tetramer binding and cytotoxic activity demonstrate that, compared to CTL from A2.1/Kb x FVB wild-type mice (A2 x FVB), CD8+ T cells from A2 x neu mice were of lower avidity for the peptides. Despite the fact that A2 x neu mice are tolerant, multiple immunizations with DC pulsed with the p369-377 or p773-782 peptides in the presence of IL-2 retarded tumor growth in A2 x neu mice, and immunizations in combination with the anti-OX40 mAb further enhanced the antitumor response. Taken together, these data indicate that low-avidity T cells for neu antigens persisting in A2 x 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 T cells in antitumor immunity and may offer an important component for vaccination immunotherapies.

Immunotherapy is an attractive strategy for cancer treatment. However, self-tolerance is one of the major mechanisms that dampen immune responses against self-tumor antigens. We have demonstrated that Her-2/neu transgenic mice (neu mice) are tolerant to neu antigens and contain only a low avidity repertoire for neu. However, this repertoire has antitumor activity. Immunizations of neu mice are capable of activating the low-avidity T cells that, at best, retard the tumor growth. To increase the efficacy of the antitumor responses in neu mice, we hypothesized that immunotherapy in combination with antiangiogenic therapy would be a more efficient strategy for tumor eradication. The rationale for using this combination was that by decreasing the growth rate of the tumor with an antiangiogenic therapy, the low-avidity repertoire of neu mice stimulated by immunotherapeutic intervention would be more effective in destroying the slow growing tumor. To test this hypothesis, we stably expressed a soluble form of the Flt-1 vascular endothelial growth factor receptor (sFlt-1) on N202.1A cells, using a retrovirus vector. Expression of sFlt-1 on N202.1A (N202-Flt) cells significantly inhibited the tumor growth compared with N202.1A parental cells. In contrast to the application of immunotherapy alone or antiangiogenic therapy alone, which delayed the tumor growth, the combination of the two therapies provided complete inhibition of tumor growth in Her-2/neu mice. These results indicate that the use of tumor targeting with immunotherapy in simultaneous combination with antiangiogenic therapy provides a more efficient strategy for the treatment of solid tumors.

The highest incidence of human cancers is seen in older people. Many parameters of immunity decrease in an ageassociated manner. These two statements are each uncontroversial. However, whether the reason for the increased frequency of cancers in the elderly is due to alterations of the immune system in the old is much more controversial. Considering that the concept of immunosurveillance against spontaneously arising tumors is itself still controversial, this is hardly surprising. However, the eYcacy of certain immunotherapy protocols in preclinical models and the much less reproducible but nonetheless occasionally highly eVective therapeutic successes achieved in humans encourages the belief that the immune system could be exploited to control tumor growth. The following “Symposium-in-Writing” was inspired by the belief that either active or adoptive immunotherapy or a combination of both will one day soon be entering standard clinical practice. Given that most studies, preclinical and clinical both, have most often been conducted in younger individuals, it is imperative to consider the eVect of age on outcome. Most obviously, the eVect of age on T cell response to vaccination will be of great importance, and also the eVect of the old host environment on the eYcacy of adoptively transferred immune eVectors needs consideration. Here, we have gathered a collection of papers which review the eVects of age on the host in terms of inXammatory status and immune responsiveness in the carcinogenic process. Salvioli et al. discuss possible reasons why centenarians are protected from cancer. This could be mediated by at least two diVerent mechanisms: low IGF-1-mediated responses and elevated production of anti-inXammatory mediators. A well-preserved p53-mediated response able to block tumorigenesis or to decrease cancer aggressiveness is also critical for cancer protection in centenarians. On the other hand, Caruso et al. discuss the eVect that inXammation has in promoting carcinogenesis. They conclude that only further knowledge of the regulation and function of inXammatory pathways in cancer will help to understand the mechanisms of tumor formation and progression, as well as to identify new targets for the reWnement of novel treatments, such as the pharmacogenomics approach. Other papers then discuss diVerent immunotherapy models where the age of the host and the age of the immune system have been speciWcally analyzed as variables aVecting the outcome of the treatment. There is a plethora of preclinical and clinical studies utilizing dendritic cell-based therapies for cancer. However, there are only a handful of studies evaluating the eVect of this type of therapy in the aging This editorial forms the introduction to the Symposium in Writing: Impact of Ageing on Cancer Immunity and Immunotherapy.

The Her-2/neu protooncogene is associated with malignant transformation and aggressive disease. Because of its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. By identifying potential HLA-A2.1-binding peptides from the Her-2/neu sequence, peptides were selected as candidate T-cell epitopes. The immunogenicity of each peptide was evaluated by priming double transgenic mice expressing both the human (hu) CD8 and HLA-A2.1 molecules with synthetic peptides corresponding to these sequences. Because of the lack of interaction between murine CD8 and HLA-A2.1, expression of huCD8 on murine cells facilitates recognition of HLA molecules on human tumor cell lines. This led to the identification of two peptides that elicit an A2-restricted CTL response, one of which has not been previously identified. Both peptidespecific CTL populations were able to specifically lyse A2.1 and Her-2/neu expressing human tumor cells originating from a variety of tissues, demonstrating the utility of this murine model in identifying peptides presented by human cells. However, several Her-2/neu peptides previously reported to be immunogenic for human CTL were found not to be immunogenic in transgenic mice. The basis for these discrepancies is discussed.

IL-9 is a cytokine that is primarily associated with mast cells and airway inflammation, however it also plays a role in T regulatory cell (Treg) survival and recruitment to tumors. We previously reported that 4-1BB treatment of Tregs inhibits both Treg function and IL-9 secretion. Neutralization of IL-9 with an anti-IL-9 antibody inhibits the suppressive function of Tregs without affecting the function of CD4+ and CD8+ T effector cells. Furthermore, the combination of intra-tumoral CpG and anti-IL-9 induced tumor rejection in BALB-neuT and MUC1 tolerant transgenic mice. Here we show that the lack of IL-9 inhibits Treg suppression of T cell proliferation, using IL-9 knockout (IL-9ko) mice. Phenotypic analysis of Treg markers showed that tumor draining lymph nodes of 4T1 bearing mice contain fewer CTLA-4+ and CD103+ Tregs, and that these also have reduced of CTLA-4 and CD103 expression. We also observed that IL-9ko mice spontaneously reject TUBO and 4T1 mammary carcinoma cells and that rejection is abrogated by CD8+ T cell depletion. Finally, preliminary studies of human CD4+ T cell proliferation revealed that IL-9 neutralization led to greater proliferation than isotype control treated cells. Taken together our results suggest that IL-9 can have an immunosuppressive function that comes into play in the tumor microenvironment, and that the blockade of IL-9 could serve as a novel strategy to perturb the function of Tregs to enhance the antitumor effect of tumor vaccines.

B cells that are destined to secrete IgE express a membrane-bound form of IgE (mIgE) on their cell surface. Thus, elimination of such mIgE-positive cells should result in the suppression of IgE production, thereby alleviating the symptoms of IgE-mediated allergy. In this study, we examined, in a model system, whether IgE-specific effector T cells can be used specifically to eradicate IgE-producing B cells. To this end, we endowed T cells with anti-IgE specificity using chimeric T cell receptors (cTCR) containing the variable region domain (Fv) of the 84.1c non-anaphylactic anti-mouse IgE monoclonal antibody (mAb). Two configurations of chimeric receptor were used: in the first, we combined the heavy and light variable region chains of 84.1c with the constant (C) regions of the TCR alpha and beta chains. The second construct consisted of a chimeric single-chain receptor (scFvR), composed of a single-chain Fv region of the 84.1c antibody and the C beta domain of the TCR. Following transfection of the cTCR or the scFvR genes into the murine MD.45 cytotoxic T cell hybridoma or the Jurkat human T cell line, functional expression of IgE-specific chimeric receptors was detected on the cell surface. The transfected cells secreted interleukin-2 upon stimulation with immobilized IgE or fixed IgE-producing hybridoma cells. Moreover, cytotoxic T cell hybridomas expressing the chimeric receptor genes specifically eliminated IgE-secreting B cells in vitro, resulting in isotype-specific suppression of IgE production.

Chronic inflammation associated with aging is termed inflamm-aging. It manifests as a rise in many proinflammatory mediators and contributes to a decline in immune function. Unregulated inflammasome activation initiates an inappropriate inflammatory response. It is particularly imperative to understand inflammasome dysregulation in the elderly (>65 yrs), since it is a likely basis of immune dysfunction. Analyses of freshly isolated memory CD4+ and CD8+ splenic T cells demonstrated that even without further activation, inflammasome genes were already significantly up-regulated in old mice compared to young, including factors critical to myeloid-derived suppressor cell (MDSC) expansion including S100a8, S100a9, IL-1β, IL-13 and IL-6. A corresponding 2-3 fold increase in CD11b+Gr1+cells was observed in the spleens of old naïve mice compared to young. Importantly, cells with the CD11b+Gr1+ phenotype from old mice, unlike young, were functionally MDSCs and suppressed T cell proliferation. Therefore, the immunosuppressive environment of the tumor-bearing host also appears to be operative in aging, even in the absence of tumor. We hypothesize that constituitive activation of inflammasome complex results in a chronic inflammatory state, altering immune responses in the elderly similarly to the tumor-bearing state. Preemptive reversal of inflammasome-induced MDSCs may enhance T cell immunosurveillance in the elderly, a population with a heightened susceptibility to malignancy.

T-regulatory cells (Tregs) create an immunosuppressive environment that reduces the efficacy of anti-tumor immunotherapy. Depletion of Tregs enhances anti-tumor immune responses, but also affects T-effector cells. Previously we showed that blockade of chemokine ligand 1 (CCL1) inhibits Treg conversion, their suppressive function, and in combination with intra-tumoral CpG-ODN results in complete rejection of tumors in BALB-NeuT tolerant mice. Most importantly, this therapeutic combination does not affect T-effector function and increases tumor-lytic CD8+NKG2D+ cells within the tumor. Here we expand on the role of blocking CCL1 in enhancing anti-tumor immunotherapy. Using CCL1 knockout (CCL1ko) mice, we found no impairment in TGFβ dependent de novo Treg conversion, however CCL1ko Tregs are less suppressive in vitro, than their wild type counterparts. CCL1ko mice treated with intratumoral CpG showed a marked reduction in tumor growth and a significant increase in tumor rejection as compared to similarly treated C57b control mice. Examination of lymphocyte populations within B16 and MucI tumors showed elevated levels of CD8+ cells. Finally the evaluation of levels of intra-tumoral cytokines and chemokines revealed elevated levels of IFNγ, CXCL9, CCL3 and CXCL10 in tumors grown in CCL1ko mice, suggesting a pro-inflammatory environment. Taken together these data support the role of CCL1as a critical factor for the maintenance of the suppressive function of Tregs.

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.

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.

Age related changes compromise T cell mediated immunity. Previous findings suggest that the aged immune T cell repertoire can be exploited to mount effective anti-tumor responses to immunogenic tumors. However, antitumor immune responses against tumor self-antigens are significantly dysregulated in the old; hence, the salient differences in the biology and function of T cells between young and old, should be elucidated. These differences fall into three categories: (i) there are lower surface levels of T cell receptor (TCR) on old CD4 and CD8 T cells, (ii) the TCR complex is cycled differently in aged T cells and (iii) the TCR complex may be disassociated in older animals. Therefore these preliminary findings support the hypothesis that aging causes a decline in T cell function. Data analyses from a T cell activation time-course microarray study show that there are large differences in the gene expression profiles of old and young naive cells in CD4 (2022 genes) and CD8 (3666 genes) subsets. These differing genetic signatures point to potentially dysregulated proteins in the old immune system. Further investigation of selected gene products using appropriate mouse models, may provide valuable preclinical information with significant therapeutic potential which could have the opportunity of being translated into clinical trials. This work was sponsored in part by a grant from AFAR.