Faculty Bibliography

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per mum(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

Activation of the Nlrc4 inflammasome results in the secretion of IL-1beta and IL-18 through caspase-1 and induction of pyroptosis. L. monocytogenes engineered to activate Nlrc4 by expression of Legionella pneumophilia flagellin (L. monocytogenes L.p.FlaA) are less immunogenic for CD8(+) T cell responses than wt L. monocytogenes. It is also known that IL-1beta orchestrates recruitment of myelomonocytic cells (MMC), which have been shown to interfere with T cell-dendritic cells (DC) interactions in splenic white pulp (WP), limiting T cell priming and protective immunity. We have further analyzed the role of MMCs in the immunogenicity of L. monocytogenes L.p.FlaA. We confirmed that MMCs infiltrate the WP between 24-48 hours in response to wt L. monocytogenes infection and that depletion of MMCs enhances CD8(+) T cell priming and protective memory. L. monocytogenes L.p.FlaA elicited accelerated recruitment of MMCs into the WP. While MMCs contribute to control of L. monocytogenes L.p.FlaA, MMC depletion did not increase immunogenicity of L.p.FlaA expressing strains. There was a significant decrease in L. monocytogenes L.p.FlaA in CD8alpha(+) DCs independent of MMCs. These findings suggest that limiting inflammasome activation is important for bacterial accumulation in CD8alpha(+) DCs, which are known to be critical for T cell response to L. monocytogenes.

A major obstacle to the treatment of Multiple Myeloma (MM) is the localization of myeloma cells to the bone marrowstroma, enabling drug resistance. The exact mechanisms of adhesion of myeloma cells to the bone marrow are not known, but adhesion molecules and chemokine signals, in particular vascular cell adhesion protein 1 (VCAM-1) and C-X-C chemokine 12 (CXCL12) which control bone marrow tropism, are thought to be the main players. Netrin-1, which acts as an axonal guidance cue, plays a role in leukocyte migration in lymph nodes and in atherosclerotic lesions, but has not been tested as a substrate for myeloma cell adhesion previously. Based on expression of the netrin-1 receptor Deleted in Colorectal Cancer (DCC) on activated human B cells, we tested the ability of myeloma cells to adhere to netrin-1, an axonal guidance cue. Using interference reflection microscopy (IRM) which employs the method of interference of light reflected from nearby surfaces to measure cell-substratum distances and cell-substratum adhesion, we assessed cell adhesion and cell spreading on substrates immobilized on glass. Here, we used this technique to assess myeloma cell adhesion and migration on various substrates and found netrin-1 to be an exceptional adhesion ligand for myeloma cells. We prepared glass substrates coated with the recombinants ligands intercellular adhesion molecule,ICAM-1(50muM), and VCAM-1(50muM), with or without chemokine ligand, CXCL12(0.1mg/mL), which have been implicated in plasma cells and myeloma cell migration, previously. We used freshly purified, fluorescently-labeled primary myeloma cells from newly diagnosed patients, prior to any treatment. Using IRM, we imaged the cell contacting the substrate in order to measure adhesion and differentiate crawling versus fluid flow movement. Based on the IRM image, we could calculate the fraction of cells in the field that were adhered to the substrate, and compared between conditions and for various patient samples. We observed that myeloma cells can adhere and migrate slowly on VCAM-1 in the presence of CXCL12, but are unable to adhere to ICAM-1 with or without chemokines. We tested myeloma cell binding to netrin-1 and saw a strong adhesion 60-90% of cells in 7 out of 9 patients samples tested. The cell spreading on netrin-1 was more than 3 times larger than on VCAM-1 with CXCL12 substrates. Expression of netrin-1 in the bone marrow has not been determined yet nor its role in MM. Heparin-like molecule, SST0001, has been tested in myeloma studies, in an attempt to interfere with heparinase activity and syndecan-1 shedding, and tumor growth. We tested pre-blocking netrin-1 substrates with heparin and observed elimination of greater than 95% of myeloma cell adhesion in all patients samples tested. Treating patients with heparin-like molecules may have additional functions, by blocking binding to netrin-1 and soluble signals that contain heparin-binding domains. Reciprocally, blocking heparin-sulfated groups with netrin-1 may block myeloma cell adhesion and can be used to targeting strategy for chemotherapeutic drugs as well

A promising strategy for cancer immunotherapy is to disrupt key pathways regulating immune tolerance, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4). However, the determinants of response to anti-CTLA-4 mAb treatment remain incompletely understood. In murine models, anti-CTLA-4 mAbs alone fail to induce effective immune responses to poorly immunogenic tumors but are successful when combined with additional interventions, including local ionizing radiation (IR) therapy. We employed an established model based on control of a mouse carcinoma cell line to study endogenous tumor-infiltrating CD8+ T lymphocytes (TILs) following treatment with the anti-CTLA-4 mAb 9H10. Alone, 9H10 monotherapy reversed the arrest of TILs with carcinoma cells in vivo. In contrast, the combination of 9H10 and IR restored MHC class I-dependent arrest. After implantation, the carcinoma cells had reduced expression of retinoic acid early inducible-1 (RAE-1), a ligand for natural killer cell group 2D (NKG2D) receptor. We found that RAE-1 expression was induced by IR in vivo and that anti-NKG2D mAb blocked the TIL arrest induced by IR/9H10 combination therapy. These results demonstrate that anti-CTLA-4 mAb therapy induces motility of TIL and that NKG2D ligation offsets this effect to enhance TILs arrest and antitumor activity.

Foxp3 activity is essential for the normal function of the immune system. Two types of regulatory T (T reg) cells express Foxp3, thymus-generated natural T reg (nT reg) cells, and peripherally generated adaptive T reg (iT reg) cells. These cell types have complementary functions. Until now, it has not been possible to distinguish iT reg from nT reg cells in vivo based solely on surface markers. We report here that Neuropilin 1 (Nrp1) is expressed at high levels by most nT reg cells; in contrast, mucosa-generated iT reg and other noninflammatory iT reg cells express low levels of Nrp1. We found that Nrp1 expression is under the control of TGF-beta. By tracing nT reg and iT reg cells, we could establish that some tumors have a very large proportion of infiltrating iT reg cells. iT reg cells obtained from highly inflammatory environments, such as the spinal cords of mice with spontaneous autoimmune encephalomyelitis (EAE) and the lungs of mice with chronic asthma, express Nrp1. In the same animals, iT reg cells in secondary lymphoid organs remain Nrp1(low). We also determined that, in spontaneous EAE, iT reg cells help to establish a chronic phase of the disease.

PROBLEM: Pregnancy is a challenge to the maternal immune system as it must defend the body against pathogens while at the same time develop immune tolerance against the fetus growing inside the uterus. Despite ex vivo techniques being used to understand these processes, in vivo techniques are missing. METHOD OF STUDY: To directly study these phenomena, we have developed a new microscope stage and surgical procedures for use in two-photon microscopy, for in vivo observation of the mouse placenta. RESULTS: These tools and surgical procedures demonstrate fetal and maternal blood flow inside the labyrinth zone of the placenta, as well as its three dimensional structure. It was also useful to identify Plasmodium chabaudi-infected red blood cells inside this labyrinth zone. CONCLUSION: We believe this technique will represent an important contribution for expanding the available knowledge concerning cell dynamics and interactions at the fetal-maternal interface.

FIVM has provided many insights into the regulation of immunity. We report the validation of an approach for visualizing murine small bowel via single- and multiphoton FIVM. Tissue damage is limited to approximately 200 mum, immediately adjacent to the incision, as confirmed by intravital PI staining. Treatment with 10 KDa dextran-FITC and 70 KDa dextran-TR confirms that perfusion is intact. Selective filtration of 10 KDa but not 70 KDa dextran from the blood indicated that kidney function is also intact. Interestingly, lamina propria vasculature is semipermeable to 10 KDa dextran. Next, reporter mice expressing egfp from the CX3CR1 locus, egfp from the FoxP3 locus, or RFP from the IL-17F locus were used to track DC subsets, FoxP3(+) Tregs, or Th17f cells, respectively. Resident cx3cr1(+/egfp) cells were sessile but actively probed the surrounding microenvironment. Both T cell populations patrol the lamina propria, but the Th17f cells migrate more rapidly than Tregs. Together, these data demonstrate intact vascular perfusion, while intravitally visualizing the mucosal surface of the small bowel. Lastly, the cx3cr1(+) DCs and T cells display activity similar to that found in steady-state, secondary lymphoid organs.

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the alpha-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.