Paola Pittoni

Invariant NKT cells sustain specific B cell responses and memory

Invariant natural killer T (iNKT) cells are innate-like lymphocytes recognizing CD1d-restricted glycolipid antigens, such as α-galactosylceramide (αGC). We assessed whether iNKT cells help B lymphocyte responses and found that mice immunized with proteins and αGC develop antibody titers 1–2 logs higher than those induced by proteins alone. Activation of iNKT cells enhances protection against infections such as influenza and elicits higher frequencies of memory B cells and higher antibody responses to booster immunizations. Protein vaccination with αGC, but not with conventional adjuvants, elicits IgG responses in mice lacking MHC class II molecules, demonstrating that iNKT cells can substitute for CD4+ T cell help to B cells. Interestingly, the decay of circulating antibodies is faster in mice lacking iNKT cells. These findings point to a homeostatic role for iNKT cells on critical features of the antibody response such as immunity and B cell memory.

Mast Cell Targeting Hampers Prostate Adenocarcinoma Development but Promotes the Occurrence of Highly Malignant Neuroendocrine Cancers

Mast cells (MC) are c-Kit-expressing cells, best known for their primary involvement in allergic reactions, but recently reappraised as important players in either cancer promotion or inhibition. Here, we assessed the role of MCs in prostate tumor development. In prostate tumors from both tumor-prone transgenic adenocarcinoma of the mouse prostate (TRAMP) mice and human patients, MCs are specifically enriched and degranulated in areas of well-differentiated (WD) adenocarcinoma but not around poorly differentiated (PD) foci that coexist in the same tumors. We derived novel TRAMP tumor cell lines, representative of WD and PD variants, and through pharmacologic stabilization or genetic ablation of MCs in recipients mice, we showed that MCs promote WD adenocarcinoma growth but are dispensable for PD tumors. WD tumors rely on MCs for matrix metalloprotease 9 (MMP-9) provision, as reconstitution of MC-deficient mice with wild-type but not MMP-9(-/-) MCs was sufficient to promote their growth. In contrast, PD tumors are MMP-9 self-competent, consistently with epithelial-to-mesenchymal transition. Such a dual source of MMP-9 was confirmed in human tumors, suggesting that MCs could be a good target for early-stage prostate cancer. Interestingly, in testing whether MC targeting could block or delay tumorigenesis in tumor-prone TRAMP mice, we observed a high incidence of early and aggressive tumors, characterized by a neuroendocrine (NE) signature and c-Kit expression. Taken together, these data underscore the contribution of MCs in tumor progression and uncover a new, opposite role of MCs in protecting against the occurrence of aggressive NE variants in prostate cancer.

Targeted Expression of Human CD1d in Transgenic Mice Reveals Independent Roles for Thymocytes and Thymic APCs in Positive and Negative Selection of Vα14i NKT Cells

CD1d-dependent invariant Vα14 (Vα14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Vα14-Jα18 TCR α-chain paired mostly with Vβ8.2 and Vβ7. The cellular requirements for thymic positive and negative selection of Vα14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Vα14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Vα14i NKT cells that proved functional when activated ex vivo with the Ag α-galactosyl ceramide. Vα14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vβ8.2. The low number of Vβ8.2+ Vα14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vβ7+ Vα14i NKT cells. Vβ8.2+, but not Vβ7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Vα14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.

Exacerbated experimental autoimmune encephalomyelitis in mast-cell-deficient Kit W-sh/W-sh mice

Mast cell (MC)-deficient c-Kit mutant KitW/W-v mice are protected against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggesting a detrimental role for MCs in this disease. To further investigate the role of MCs in EAE, we took advantage of a recently characterized model of MC deficiency, KitW-sh/W-sh. Surprisingly, we observed that myelin oligodendrocyte glycoprotein (MOG)35–55-induced chronic EAE was exacerbated in KitW-sh/W-sh compared with Kit+/+ mice. KitW-sh/W-sh mice showed more inflammatory foci in the central nervous system (CNS) and increased T-cell response against myelin. To understand whether the discrepant results obtained in KitW-sh/W-sh and in KitW/W-v mice were because of the different immunization protocols, we induced EAE in these two strains with varying doses of MOG35–55 and adjuvants. Although KitW-sh/W-sh mice exhibited exacerbated EAE under all immunization protocols, KitW/W-v mice were protected from EAE only when immunized with high, but not low, doses of antigen and adjuvants. KitW-sh/W-sh mice reconstituted systemically, but not in the CNS, with bone marrow-derived MCs still developed exacerbated EAE, indicating that protection from disease could be exerted by MCs mainly in the CNS, and/or by other cells possibly dysregulated in KitW-sh/W-sh mice. In summary, these data suggest to reconsider MC contribution to EAE, taking into account the variables of using different experimental models and immunization protocols.

A non-redundant role for OX40 in the competitive fitness of Treg in response to IL-2

OX40 stimulation is known to enhance activation of effector T cells and to inhibit induction and suppressive function of Treg. Here we uncovered a novel role of OX40 in sustaining Treg competitive fitness in vivo, during repopulation of lymphopenic hosts and reconstitution of BM chimeras. Defective expansion of OX40‐null Treg diminished their ability to suppress inflammation in a model of lymphopenia‐driven colitis. OX40‐mediated promotion of Treg fitness spanned beyond lymphopenic environments, as endogenous Treg in OX40‐null mice showed decreased accumulation during thymic development, enhanced susceptibility to antibody‐mediated depletion and defective turnover following thymectomy. In vitro, OX40‐deficient Treg were found to be intrinsically hyporesponsive to IL‐2, in terms of Stat5 phosphorylation and proliferation, according to elevated SOCS1 content and reduced miR155 expression. Therefore, OX40 is a key factor in shaping Treg sensitivity to IL‐2 and promoting their proliferation and survival, toward accurate immune regulation.

Intratumor OX40 stimulation inhibits IRF1 expression and IL‐10 production by Treg cells while enhancing CD40L expression by effector memory T cells

Treg cells maintain the tumor microenvironment in an immunosuppressive state preventing an effective anti‐tumor immune response. A possible strategy to overcome Treg‐cell suppression focuses on OX40, a costimulatory molecule expressed constitutively by Treg cells while being induced in activated effector T cells. OX40 stimulation, by the agonist mAb OX86, inhibits Treg‐cell suppression and boosts effector T‐cell activation. Here we uncover the mechanisms underlying the therapeutic activity of OX86 treatment dissecting its distinct effects on Treg and on effector memory T (Tem) cells, the most abundant CD4+ populations strongly expressing OX40 at the tumor site. In response to OX86, tumor‐infiltrating Treg cells produced significantly less interleukin 10 (IL‐10), possibly in relation to a decrease in the transcription factor interferon regulatory factor 1 (IRF1). Tem cells responded to OX86 by upregulating surface CD40L expression, providing a licensing signal to DCs. The CD40L/CD40 axis was required for Tem‐cell‐mediated in vitro DC maturation and in vivo DC migration. Accordingly, OX86 treatment was no longer therapeutic in CD40 KO mice. In conclusion, following OX40 stimulation, blockade of Treg‐cell suppression and enhancement of the Tem‐cell adjuvant effect both concurred to free DCs from immunosuppression and activate the immune response against the tumor.

The Dark Side of Mast Cell–Targeted Therapy in Prostate Cancer

Tumor development requires accomplices among white blood cells. Other than macrophages, mast cells have been observed to support the outgrowth of certain neoplasias because of their proangiogenic properties. In some tumor settings, however, mast cells may have a protective role, exerted by their proinflammatory mediators. In prostate cancer, no conclusive data on mast cell function were available. Here, we discuss recent work on the role of mast cells in mouse and human prostate cancer, showing that mast cells can behave alternatively as dangerous promoters, innocent bystanders, or essential guardians of tumors, according to the stage and origin of transformed cells. In particular, mast cells are essential for the outgrowth of early-stage tumors due to their matrix metalloproteinase-9 production, become dispensable in advanced-stage, post-epithelial-to-mesenchymal transition, and are protective against neuroendocrine prostate tumor variants. The common expression of c-Kit by mast cells and neuroendocrine clones suggests a possible competition for the ligand Stem cell factor and offers the chance of curing early-stage disease while preventing neuroendocrine tumors using c-Kit-targeted therapy. This review discusses the implications of these findings on the advocated mast cell-targeted cancer therapy and considers future directions in the study of mast cells and their interactions with other c-Kit-expressing cells.

Phage display-derived recombinant antibodies with TCR-like specificity against α-galactosylceramide and its analogues in complex with human CD1d molecules

The glycolipid α‐galactosylceramide (α‐GalCer) is a potent activator of invariant natural killer T (iNKT) cells and has been shown to be an effective agent against cancer, infections and autoimmune diseases. The effectiveness of α‐GalCer and its alkyl chain analogues depends on efficient loading and presentation by the antigen‐presenting molecule CD1d. To monitor the ability of CD1d to present the glycolipids, we have used a phage display strategy to generate recombinant antibodies with T cell receptor‐like (TCRL) specificity against the human CD1d (hCD1d)‐α‐GalCer complex. These Fab fragments were able to detect specifically hCD1d‐α‐GalCer complexes in cell‐free systems such as surface plasmon resonance and ELISA, as well as on the surface of hCD1d+ antigen‐presenting cells (APC) by flow cytometry and immunofluorescence microscopy, the latter of which could also detect intracellular complexes. We show that our TCRL antibodies can stain dendritic cells from CD11c‐hCD1d‐transgenic mice administered in vivo with α‐GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of α‐GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the α‐GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of α‐GalCer is improved with longer incubation times on professional APC. This reagent will be essential for assessing the loading and presenting capabilities of hCD1d of α‐GalCer and its analogues.

Functional Education of Invariant NKT Cells by Dendritic Cell Tuning of SHP-1

Invariant NKT (iNKT) cells play key roles in host defense by recognizing lipid Ags presented by CD1d. iNKT cells are activated by bacterial-derived lipids and are also strongly autoreactive toward self-lipids. iNKT cell responsiveness must be regulated to maintain effective host defense while preventing uncontrolled stimulation and potential autoimmunity. CD1d-expressing thymocytes support iNKT cell development, but thymocyte-restricted expression of CD1d gives rise to Ag hyperresponsive iNKT cells. We hypothesized that iNKT cells require functional education by CD1d+ cells other than thymocytes to set their correct responsiveness. In mice that expressed CD1d only on thymocytes, hyperresponsive iNKT cells in the periphery expressed significantly reduced levels of tyrosine phosphatase SHP-1, a negative regulator of TCR signaling. Accordingly, heterozygous SHP-1 mutant mice displaying reduced SHP-1 expression developed a comparable population of Ag hyperresponsive iNKT cells. Restoring nonthymocyte CD1d expression in transgenic mice normalized SHP-1 expression and iNKT cell reactivity. Radiation chimeras revealed that CD1d+ dendritic cells supported iNKT cell upregulation of SHP-1 and decreased responsiveness after thymic emigration. Hence, dendritic cells functionally educate iNKT cells by tuning SHP-1 expression to limit reactivity.

Mast Cells and Immune Response in Cancer

Mast cells are the main promoters of allergic reactions, but they intervene in many other physiological and pathological conditions. For many decades, their involvement in tumor biology has been recognized, but a series of recent studies has greatly contributed to clarifying some important aspects regarding the specific involvement of mast cells in the tumor microenvironment. Data from human samples indicate mast cells as associated to either better or worst prognosis depending on tumor types and stages. Results obtained in mouse models have demonstrated that mast cells influence tumor progression, thanks to their ability to promote angiogenesis, modulate antitumor immune responses, and regulate tumor growth. All these properties are mediated by a huge variety of receptors and effector molecules, which make mast cells an extremely plastic and eclectic immune cell. In light of their pro-tumor role, mast cells may be targeted thanks to old drugs used to treat allergic disease, or to potent tyrosine kinase inhibitors acting on the c-Kit receptor.