Transcriptional activation of Jun and Fos members of the AP-1 complex is a conserved signature of immune aging that contributes to inflammaging.

Diverse mouse strains have different health and life spans, mimicking the diversity among humans. To capture conserved aging signatures, we studied long-lived C57BL/6J and short-lived NZO/HILtJ mouse strains by profiling transcriptomes and epigenomes of immune cells from peripheral blood and the spleen from young and old mice. Transcriptional activation of the AP-1 transcription factor complex, particularly Fos, Junb, and Jun genes, was the most significant and conserved aging signature across tissues and strains. ATAC-seq data analyses showed that the chromatin around these genes was more accessible with age and there were significantly more binding sites for these TFs with age across all studied tissues, targeting pro-inflammatory molecules including Il6. Age-related increases in binding sites of JUN and FOS factors were also conserved in human peripheral blood ATAC-seq data. Single-cell RNA-seq data from the mouse aging cell atlas Tabula Muris Senis showed that the expression of these genes increased with age in B, T, NK cells, and macrophages, with macrophages from old mice expressing these molecules more abundantly than other cells. Functional data showed that upon myeloid cell activation via poly(I:C), the levels of JUN protein and its binding activity increased more significantly in spleen cells from old compared to young mice. In addition, upon activation, old cells produced more IL6 compared to young cells. In sum, we showed that the aging-related transcriptional activation of Jun and Fos family members in AP-1 complex is conserved across immune tissues and long- and short-living mouse strains, possibly contributing to increased inflammation with age.

Concomitant inhibition of PPARγ and mTORC1 induces the differentiation of human monocytes into highly immunogenic dendritic cells.

Monocytes can differentiate into macrophages (Mo-Macs) or dendritic cells (Mo-DCs). The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the differentiation of monocytes into Mo-Macs, while the combination of GM-CSF/interleukin (IL)-4 is widely used to generate Mo-DCs for clinical applications and to study human DC biology. Here, we report that pharmacological inhibition of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the presence of GM-CSF and the absence of IL-4 induces monocyte differentiation into Mo-DCs. Remarkably, we find that simultaneous inhibition of PPARγ and the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) induces the differentiation of Mo-DCs with stronger phenotypic stability, superior immunogenicity, and a transcriptional profile characterized by a strong type I interferon (IFN) signature, a lower expression of a large set of tolerogenic genes, and the differential expression of several transcription factors compared with GM-CSF/IL-4 Mo-DCs. Our findings uncover a pathway that tailors Mo-DC differentiation with potential implications in the fields of DC vaccination and cancer immunotherapy.

The Th1/Tfh-like biased responses elicited by the rASP-1 innate adjuvant are dependent on TRIF and Type I IFN receptor pathways.

Ov-ASP-1 (rASP-1), a parasite-derived protein secreted by the helminth Onchocerca volvulus, is an adjuvant which enhances the potency of the influenza trivalent vaccine (IIV3), even when used with 40-fold less IIV3. This study is aimed to provide a deeper insight into the molecular networks that underline the adjuvanticity of rASP-1. Here we show that rASP-1 stimulates mouse CD11c+ bone marrow-derived dendritic (BMDCs) to secrete elevated levels of IL-12p40, TNF-α, IP-10 and IFN-ß in a TRIF-dependent but MyD88-independent manner. rASP-1-activated BMDCs promoted the differentiation of naïve CD4+ T cells into Th1 cells (IFN-γ+) that was TRIF- and type I interferon receptor (IFNAR)-dependent, and into Tfh-like cells (IL21+) and Tfh1 (IFN-γ+ IL21+) that were TRIF-, MyD88- and IFNAR-dependent. rASP-1-activated BMDCs promoted the differentiation of naïve CD4+ T cells into Th17 (IL-17+) cells only when the MyD88 pathway was inhibited. Importantly, rASP-1-activated human blood cDCs expressed upregulated genes that are associated with DC maturation, type I IFN and type II IFN signaling, as well as TLR4-TRIF dependent signaling. These activated cDCs promoted the differentiation of naïve human CD4+ T cells into Th1, Tfh-like and Th17 cells. Our data thus confirms that the rASP-1 is a potent innate adjuvant that polarizes the adaptive T cell responses to Th1/Tfh1 in both mouse and human DCs. Notably, the rASP-1-adjuvanted IIV3 vaccine elicited protection of mice from a lethal H1N1 infection that is also dependent on the TLR4-TRIF axis and IFNAR signaling pathway, as well as on its ability to induce anti-IIV3 antibody production.

Sestrins induce natural killer function in senescent-like CD8+ T cells.

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8+ T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27-CD28-CD8+ T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27-CD28-CD8+ T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27-CD28-CD8+ T cells to acquire a broad-spectrum, innate-like killing activity.

The importance of cellular internalization of antibody-targeted carbon nanotubes in the photothermal ablation of breast cancer cells.

Single-walled carbon nanotubes (CNTs) convert absorbed near infrared (NIR) light into heat. The use of CNTs in the NIR-mediated photothermal ablation of tumor cells is attractive because the penetration of NIR light through normal tissues is optimal and the side effects are minimal. Targeted thermal ablation with minimal collateral damage can be achieved by using CNTs attached to tumor-specific monoclonal antibodies (MAbs). However, the role that the cellular internalization of CNTs plays in the subsequent sensitivity of the target cells to NIR-mediated photothermal ablation remains undefined. To address this issue, we used CNTs covalently coupled to an anti-Her2 or a control MAb and tested their ability to bind, internalize, and photothermally ablate Her2(+) but not Her2(-) breast cancer cell lines. Using flow cytometry, immunofluorescence, and confocal Raman microscopy, we observed the gradual time-dependent receptor-mediated endocytosis of anti-Her2-CNTs whereas a control MAb-CNT conjugate did not bind to the cells. Most importantly, the Her2(+) cells that internalized the MAb-CNTs were more sensitive to NIR-mediated photothermal damage than cells that could bind to, but not internalize the MAb-CNTs. These results suggest that both the targeting and internalization of MAb-CNTs might result in the most effective thermal ablation of tumor cells following their exposure to NIR light.

Specific thermal ablation of tumor cells using single-walled carbon nanotubes targeted by covalently-coupled monoclonal antibodies.

CD22 is broadly expressed on human B cell lymphomas. Monoclonal anti-CD22 antibodies alone, or coupled to toxins, have been used to selectively target these tumors both in SCID mice with xenografted human lymphoma cell lines and in patients with B cell lymphomas. Single-walled carbon nanotubes (CNTs) attached to antibodies or peptides represent another approach to targeting cancer cells. CNTs convert absorbed near-infrared (NIR) light to heat, which can thermally ablate cells that have bound the CNTs. We have previously demonstrated that monoclonal antibodies (MAbs) noncovalently coupled to CNTs can specifically target and kill cells in vitro. Here, we describe the preparation of conjugates in which the MAbs are covalently conjugated to the CNTs. The specificity of both the binding and NIR-mediated killing of the tumor cells by the MAb-CNTs is demonstrated by using CD22+CD25- Daudi cells, CD22-CD25+ phytohemagglutinin-activated normal human peripheral blood mononuclear cells, and CNTs covalently modified with either anti-CD22 or anti-CD25. We further demonstrate that the stability and specificity of the MAb-CNT conjugates are preserved following incubation in either sodium dodecyl sulfate or mouse serum, indicating that they should be stable for in vivo use.

Thermal ablation of tumor cells with antibody-functionalized single-walled carbon nanotubes.

Single-walled carbon nanotubes (CNTs) emit heat when they absorb energy from near-infrared (NIR) light. Tissue is relatively transparent to NIR, which suggests that targeting CNTs to tumor cells, followed by noninvasive exposure to NIR light, will ablate tumors within the range of NIR. In this study, we demonstrate the specific binding of antibody-coupled CNTs to tumor cells in vitro, followed by their highly specific ablation with NIR light. Biotinylated polar lipids were used to prepare stable, biocompatible, noncytotoxic CNT dispersions that were then attached to one of two different neutralite avidin-derivatized mAbs directed against either human CD22 or CD25. CD22(+)CD25(-) Daudi cells bound only CNTs coupled to the anti-CD22 mAb; CD22(-)CD25(+) activated peripheral blood mononuclear cells bound only to the CNTs coupled to the anti-CD25 mAb. Most importantly, only the specifically targeted cells were killed after exposure to NIR light.

Cancer dormancy: from mice to man.

In this review, we focused on our studies of cancer dormancy in a murine B cell lymphoma and human breast cancer. Lifelong dormancy was induced in syngeneic mice by prior immunization to the idiotype of the tumor cell (TC) Ig before TC challenge. The mice maintained approximately 10(6) lymphoma cells in their spleen throughout their lifetime despite replication of the TCs at a reduced rate. Recurrences occurred randomly. Because of the balance between replication and cell death, we hypothesized that a similar balance might occur in long-term survivors of breast cancer when the risk of recurrences is very low. We developed a sensitive assay for circulating tumor cells (CTCs) which none were found in normal age-matched women. One third of patients, 7-22 years after mastectomy and without any evidence of disease, had CTCs. The half-life of these CTCs could be deduced from other studies as probably 2-3 hours. Hence, there was a precise balance between replication of TCs (presumably from micrometastases) and cell death. Therefore, a major population of clinically cured breast cancer patients have a chronic disease controlled by their own physiological mechanisms. We speculate on underlying mechanisms based both on studies in experimental models and clinical trials.

Mathematical models of the fate of lymphoma B cells after antigen receptor ligation with specific antibodies.

We formulate models of the mechanism(s) by which B cell lymphoma cells stimulated with an antibody specific to the B cell receptor (IgM) become quiescent or apoptotic. In particular, we aim to reproduce experimental results by Marches et al. according to which the fate of the targeted cells (Daudi) depends on the levels of expression of p21(Waf1) (p21) cell-cycle inhibitor. A simple model is formulated in which the basic ingredients are p21 and caspase activity, and their mutual inhibition. We show that this model does not reproduce the experimental results and that further refinement is needed. A second model successfully reproduces the experimental observations, for a given set of parameter values, indicating a critical role for Myc in the fate decision process. We use bifurcation analysis and objective sensitivity analysis to assess the robustness of our results. Importantly, this analysis yields experimentally testable predictions on the role of Myc, which could have therapeutic implications.

Enhancement of the p27Kip1-mediated antiproliferative effect of trastuzumab (Herceptin) on HER2-overexpressing tumor cells.

The oncogenic activity of the overexpressed HER2 tyrosine kinase receptor requires its localization in the plasma membrane. The antitumor effect of anti-HER2 antibodies (Abs) is mainly dependent on receptor downregulation and comprises p27Kip1-mediated G1 cell cycle arrest. However, one major limitation of anti-HER2 therapy is the reversibility of tumor growth inhibition after discontinuation of treatment caused by the mitogenic signaling associated with cell surface receptor re-expression. We found that the level of p27Kip1 upregulation, inhibition of Cdk2 activity and magnitude of G1 arrest induced by the humanized Ab trastuzumab (Herceptin, HCT) on BT474 and SKBr3 HER2-overexpressing breast cancer cells correlates with the level of cell surface receptor. Thus, continuous exposure of cells to HCT for 72 hr results in downregulation of the cell surface receptor and a concurrent increase in the level of p27Kip1 protein. Discontinuation of Ab exposure after the first 8 hr results in failure to upregulate p27Kip1 and arrest of cell cycle progression. We show that the lysosomotropic amine chloroquine (CQ) augments receptor internalization in HER2-overexpressing cells either pretreated or continuously treated with HCT and leads to an increased and sustained inhibitory effect. The enhanced CQ-dependent loss of functional HER2 from the cell surface resulted in sustained inactivation of the serine/threonine kinase Akt, upregulation of p27Kip1 protein and inhibition of cyclin E/Cdk2 activity. Potentiation of the inhibitory effect of HCT by CQ was directly related to loss of HER2 from the plasma membrane since prevention of Ab-mediated receptor endocytosis by engagement of the receptor with immobilized HCT abrogated the effect of CQ.

An anti-CD19 antibody inhibits the interaction between P-glycoprotein (P-gp) and CD19, causes P-gp to translocate out of lipid rafts, and chemosensitizes a multidrug-resistant (MDR) lymphoma cell line.

We have previously demonstrated that an anti-CD19 monoclonal antibody (MAb; HD37) inhibits the function of the P-glycoprotein (P-gp) pump in a multidrug-resistant (MDR) B-lymphoma cell line, Namalwa/MDR1, and that this effect is not due to the recognition of a cross-reactive epitope on P-gp. In this study, we have used the same cell line to define the mechanisms responsible for the effect of HD37 on the P-gp pump. Using fluorescence resonance energy transfer (FRET), we show that CD19 and P-gp are constitutively associated in cells. In the absence of treatment with anti-CD19, 40% of P-gp molecules expressed by Namalwa/MDR1 cells reside in the low-density lipid (ie, cholesterol-rich) microdomains (lipid rafts). Following treatment of the cells with HD37 and disruption of the interactions between P-gp and CD19, P-gp translocated out of lipid rafts and CD19 translocated into lipid rafts. The effect of chemosensitization on Namalwa/MDR1 cells was specific for CD19; an anti-CD22 MAb had no such effect, although the cells express CD22. These results suggest that anti-CD19 might chemosensitize P-gp(+) cells by interfering with interactions between CD19 and P-gp, rapidly resulting in the translocation of P-gp into a compartment on the plasma membrane where it is no longer active.

Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved antigrowth activity of a human breast cancer cell line in vitro and in vivo.

Her-2 (p185(erbB-2)) is a transmembrane tyrosine kinase receptor, which is encoded by the Her-2/neu proto-oncogene. Her-2 is overexpressed on 30% of highly malignant breast cancers. Monoclonal antibodies (MAbs) against Her-2 inhibit the growth of Her-2-overexpressing tumor cells and this occurs by a variety of mechanisms. One such MAb, Herceptin (Trastuzumab), has been approved for human use. We have generated a panel of murine anti-Her-2 MAbs against nine different epitopes on the extracellular domain of Her-2 and have evaluated the antitumor activity of three of these MAbs alone and in combination, both in vitro and in vivo. We found that MAbs (against different epitopes) make a highly effective mixture, which was more effective than the individual MAbs in treating s.c. tumor nodules of BT474 cells in SCID mice. In vitro, the MAb mixture was also more effective than the single MAbs in inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, inhibiting cell growth and inducing apoptosis, and inhibiting the secretion of vascular endothelial growth factor. Taken together, these activities might explain the superior performance of the MAb mixture in vivo.