CD3xCD19 DART molecule treatment induces non-apoptotic killing and is efficient against high-risk chemotherapy and venetoclax-resistant chronic lymphocytic leukemia cells.

BACKGROUND: Bispecific antibodies are promising new therapeutics in B cell malignancies. Whether they lead to potent T cell activation despite described T cell dysfunction in chronic lymphocytic leukemia (CLL), and are able to effectively target high-risk or venetoclax-resistant samples, is currently unknown. METHODS: CD19+ cell lines or primary (high-risk) CLL were cocultured in vitro with healthy donor (HD) or CLL-derived T cells in the presence of a CD3xCD19 dual affinity retargeting molecule (CD3xCD19 DART). Cell cytotoxicity, T cell activation, proliferation and effector molecule production were analyzed using flow cytometry. RESULTS: Here, we report that a bispecific CD3xCD19 DART mediates efficient killing by HD T cells of CD19+ cell-lines and primary CLL cells, regardless of immunoglobulin heavy chain variable region (IGHV) mutational status TP53 status or chemotherapy, ibrutinib or venetoclax sensitivity. Whereas TCR stimulation of CLL-derived T cells resulted in dysfunctional T cell activation and proliferation, treatment with CD3xCD19 DART led to a similar activation profile in CLL-derived and HD-derived T cells. Consistently, co-culture of CLL derived T cells with JeKo-1 or CLL cells in the presence of CD3xCD19 DART resulted in significant cytotoxicity by both CD4+ and CD8+ T cells. On stimulation of CLL cells with CD40L, CLL cells become resistant to the specific inhibitor of anti-apoptotic Bcl-2 protein venetoclax, due to upregulation of Bcl-2 family members such as Bcl-XL. Nevertheless, CD40L stimulated CLL cells were as efficiently lysed on CD3xCD19 DART treatment as unstimulated CLL cells. Further examination of the mechanism of CD3xCD19 DART mediated killing showed that lysis was dependent on granules, but was independent of BAX/BAK or caspase activity, indicating non-apoptotic cell death. CONCLUSIONS: These data show that CD3xCD19 DART in CLL leads to robust T cell activation and lysis of high-risk venetoclax resistant CLL cells through a non-apoptotic mechanism.

Correction: Dissecting the Autocrine and Paracrine Roles of the CCR2-CCL2 Axis in Tumor Survival and Angiogenesis.

[This corrects the article DOI: 10.1371/journal.pone.0028305.].

Self-reactive IgE exacerbates interferon responses associated with autoimmunity.

Canonically, immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE), IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs), a type of cell of the immune system linked to viral defense, which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE, followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses.

ß-mannosylceramide activates type I natural killer t cells to induce tumor immunity without inducing long-term functional anergy.

PURPOSE: Most studies characterizing antitumor properties of invariant natural killer T (iNKT) cells have used the agonist, α-galactosylceramide (α-GalCer). However, α-GalCer induces strong, long-lasting anergy of iNKT cells, which could be a major detriment for clinical therapy. A novel iNKT cell agonist, ß-mannosylceramide (ß-ManCer), induces strong antitumor immunity through a mechanism distinct from that of α-GalCer. The objective of this study was to determine whether ß-ManCer induces anergy of iNKT cells. EXPERIMENTAL DESIGN: Induction of anergy was determined by ex vivo analysis of splenocytes from mice pretreated with iNKT cell agonists as well as in the CT26 lung metastasis in vivo tumor model. RESULTS: ß-ManCer activated iNKT cells without inducing long-term anergy. The transience of anergy induction correlated with a shortened duration of PD-1 upregulation on iNKT cells activated with ß-ManCer, compared with α-GalCer. Moreover, whereas mice pretreated with α-GalCer were unable to respond to a second glycolipid stimulation to induce tumor protection for up to 2 months, mice pretreated with ß-ManCer were protected from tumors by a second stimulation equivalently to vehicle-treated mice. CONCLUSIONS: The lack of long-term functional anergy induced by ß-ManCer, which allows for a second dose to still give therapeutic benefit, suggests the strong potential for this iNKT cell agonist to succeed in settings where α-GalCer has failed.

Balance is a key for happiness.

Eliminating one immunosuppressive mechanism is rarely sufficient to overcome cancer. One of reasons underlying this fact is that whether regulatory T cells (Tregs) or type II natural killer T (NKT) cells dominate immunosuppression depends on the mutual interactions between the latter and their type I counterparts. Thus, the balance among three immunomodulatory cell types dictates whether eliminating Tregs relieves or not immunosuppression.

Delicate balance among three types of T cells in concurrent regulation of tumor immunity.

The nature of the regulatory cell types that dominate in any given tumor is not understood at present. Here, we addressed this question for regulatory T cells (Treg) and type II natural killer T (NKT) cells in syngeneic models of colorectal and renal cancer. In mice with both type I and II NKT cells, or in mice with neither type of NKT cell, Treg depletion was sufficient to protect against tumor outgrowth. Surprisingly, in mice lacking only type I NKT cells, Treg blockade was insufficient for protection. Thus, we hypothesized that type II NKT cells may be neutralized by type I NKT cells, leaving Tregs as the primary suppressor, whereas in mice lacking type I NKT cells, unopposed type II NKT cells could suppress tumor immunity even when Tregs were blocked. We confirmed this hypothesis in 3 ways by reconstituting type I NKT cells as well as selectively blocking or activating type II NKT cells with antibody or the agonist sulfatide, respectively. In this manner, we showed that blockade of both type II NKT cells and Tregs is necessary to abrogate suppression of tumor immunity, but a third cell, the type I NKT cell, determines the balance between these regulatory mechanisms. As patients with cancer often have deficient type I NKT cell function, managing this delicate balance among 3 T-cell subsets may be critical for the success of immunotherapy for human cancer.

Dissecting the autocrine and paracrine roles of the CCR2-CCL2 axis in tumor survival and angiogenesis.

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX3CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2-/-, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX3CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX3CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx3cr1(gfp)), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX3CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.

Mouse and human iNKT cell agonist ß-mannosylceramide reveals a distinct mechanism of tumor immunity.

Type 1 or invariant NKT (iNKT) cell agonists, epitomized by α-galactosylceramide, protect against cancer largely by IFN-γ-dependent mechanisms. Here we describe what we believe to be a novel IFN-γ-independent mechanism induced by ß-mannosylceramide, which also defines a potentially new class of iNKT cell agonist, with an unusual ß-linked sugar. Like α-galactosylceramide, ß-mannosylceramide directly activates iNKT cells from both mice and humans. In contrast to α-galactosylceramide, protection by ß-mannosylceramide was completely dependent on NOS and TNF-α, neither of which was required to achieve protection with α-galactosylceramide. Moreover, at doses too low for either alone to protect, ß-mannosylceramide synergized with α-galactosylceramide to protect mice against tumors. These results suggest that treatment with ß-mannosylceramide provides a distinct mechanism of tumor protection that may allow efficacy where other agonists have failed. Furthermore, the ability of ß-mannosylceramide to synergize with α-galactosylceramide suggests treatment with this class of iNKT agonist may provide protection against tumors in humans.

Predominant expression of CCL2 at the tumor site of prostate cancer patients directs a selective loss of immunological tolerance to CCL2 that could be amplified in a beneficial manner.

We have previously shown that, during inflammatory autoimmune diseases in humans, the immune system develops a neutralizing auto-Ab-based response to a very limited number of inflammatory mediators, and that amplification of each response could be beneficial for the host. Our working hypothesis has been that this selective breakdown of immunological tolerance is due to a predominant expression of an inflammatory mediator at an immune-restricted site undergoing a destructive process. All three conditions also take place in cancer diseases. In this study, we delineate this hypothesis for the first time in a human cancer disease and then explore its clinical implications. We show that in primary tumor sections of prostate cancer subjects, CCL2 is predominantly expressed at the tumor site over other chemokines that have been associated with tumor development, including: CXCL12, CXCL10, CXCL8, CCL3, and CCL5. Subsequently, the immune response selectivity mounts an Ab-based response to CCL2. These Abs are neutralizing Abs. These findings hold diagnostic and therapeutic implications. The current diagnosis of prostate cancer is based on prostate-specific Ag measurements that do not distinguish benign hypertrophy from malignancy. We show in this study that development of anti-CCL2 Abs is selective to the malignant stage. From a clinically oriented perspective, we show, in an experimental model of the disease, that DNA-based amplification of this response suppresses disease, which has implications for a novel way of therapy in humans.

A novel recombinant fusion protein encoding a 20-amino acid residue of the third extracellular (E3) domain of CCR2 neutralizes the biological activity of CCL2.

CCL2 is a key CC chemokine that has been implicated in a variety of inflammatory autoimmune diseases and in tumor progression and it is therefore an important target for therapeutic intervention in these diseases. Soluble receptor-based therapy is a known approach for neutralizing the in vivo functions of soluble mediators. Owing to the complexity of seven-transmembrane G protein-coupled receptors, efforts to generate neutralizing soluble chemokine receptors have so far failed. We developed a strategy that is based on the generation of short recombinant proteins encoding different segments of a G protein-coupled receptor, and tested the ability of each of them to bind and neutralize its target chemokine. We show that a fusion protein comprised of as few as 20 aa of the third extracellular (E3) domain of the CCL2 receptor, stabilized by the IgG H chain Fc domain (E3-IgG or BL-2030), selectively binds CCL2 and CCL16 and effectively neutralizes their biological activities. More importantly, E3-IgG (BL-2030) could effectively suppress the in vivo biological activity of CCL2, attenuating ongoing experimental autoimmune encephalomyelitis, as well as the development of human prostate tumor in SCID mice.