Cell Receptor Ligands Research Articles

Preclinical evaluation of recombinant T cell receptor ligand RTL1000 as a therapeutic agent in ischemic stroke.

Recombinant T cell Receptor Ligand 1000 (RTL1000), a partial human major histocompatibility complex (MHC) molecule coupled to a human myelin peptide, reduces infarct size after experimental stroke in HLA-DRB1*1502 transgenic (DR2-Tg) mice. In this study, we characterized the therapeutic time window of opportunity for RTL1000; we explored the efficacy of a single dose of RTL1000 administration and determined if RTL1000 affords long-term neurobehavioral functional improvement after ischemic stroke. Male DR2-Tg mice underwent 60min of intraluminal reversible middle cerebral artery occlusion (MCAO). RTL1000 or vehicle was injected 4, 6, or 8h after MCAO, followed by three daily injections. In the single-dose study, one-time injection of RTL1000 was applied 4h after MCAO. Cortical, striatal, and hemispheric infarct sizes were measured 24 or 96h after stroke. Behavioral testing, including neuroscore evaluation, open field, paw preference, and novel object recognition, was performed up to 28days after stroke. Our data showed that RTL1000 significantly reduced the infarct size 96h after MCAO when the first injection was given at 4 and 6h, but not 8h, after the onset of stroke. A single dose of 400 or 100μg RTL1000 also significantly reduced the infarct size 24h after MCAO. Behavioral testing showed that RTL1000 treatment used 4h after MCAO improved long-term cognitive outcome 28days after stroke. Taken together, RTL1000 protects against acute injury if applied within a 6-h time window and improves long-term functional recovery after experimental stroke in DR2-Tg mice.

Abstract LB-268: Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignant tumor of the bone in pediatric patients. Lungs are the most common site of metastasis. Patients with recurrent and metastatic OS are resistant to chemotherapy and have very poor survival rates. Our goal is to find an alternate more effective therapy for OS lung metastasis. Allogeneic natural killer (NK) cells are emerging as a promising treatment strategy for cancers. Although data from our lab and other labs show that OS cells are susceptible to NK cell cytotoxicity, NK cell therapy by itself may not be effective enough to cure OS since tumor cells can escape immunotherapy. One way to augment cytotoxicity of NK cell is using HDAC inhibitors (HDACi). Several studies have shown that HDACis (including entinostat) sensitize tumor cells for cytotoxic effects of NK cells mostly by inducing expression of ligands for NK cells on tumor cells. In this study we sought to investigate whether entinostat would augment the cytotoxic effects NK cells on OS cells. Methods: In our in vitro study we investigated whether entinostat would increase expression of ligands for NK cell receptors on OS cells and as a result would enhance OS cells’ susceptibility to the cytotoxic effects of NK cells. Since the combination of NK cells and entinostat will be used in our in vivo study (a mouse model with OS lung metastasis) we determined the effect of the drug on NK cell viability, receptor expression, and cytotoxicity. NK cells were isolated from healthy donor buffy coats and were expanded ex vivo for 4 weeks in RPMI 1640 media supplemented with artificial antigen-presenting cells (K562) and IL-2. Results: We demonstrated that 2μM entinostat (≤ IC50) for 48hr upregulates expression of NK cell ligands (ULBP1, ULBP2/5/6, and MIC A/B) on OS cell lines (LM7, KRIB, CCHOSD, and CCHOSO). Upregulated ligands on OS cells are stable for at least 24hr after drug removal from the cultured media. Moreover, entinostat treatment increased susceptibility of OS cell lines to cytotoxic killing by NK cells. By using blocking antibodies for NK receptors we confirmed that NK cell cytotoxicity toward OS cells is dependent on receptor and ligand interaction. Treatment of NK cells with up to 2μM entinostat did not affect the viability of NK cells. This drug did not affect the expression of NK cell receptors (NKG2D, NKp30, NKp44, NKp46, and DNAM-1) within the first 24hr; however, after 48hr 1μM and 2μM of the drug decreased expression of receptors except DNAM-1. Pre-treatment of NK cells with entinostat did not decrease cytotoxicity of NK cell to OS cell lines. Conclusion: Our results demonstrate that entinostat immunosensitizes OS cells to NK cell lysis by inducing upregulation of ligands for NK cells on OS cells. We also show that up to 2μM treatment of entinostat for 24 hr does not have any adverse effect on NK cells. These results tell us the time point at which to administer NK cells and entinostat in combination to mice in our in vivo. These results support our hypothesis, saying that combining entinostat with NK cell therapy would augment the cytotoxicity effect of NK cells on osteosarcoma lung metastasis. Citation Format: Simin Kiany, Ling Yu, Michele Guindani, Jimin Wu, John Stewart, Eugenie S Kleinerman. Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-268. doi:10.1158/1538-7445.AM2014-LB-268

The role of microRNAs in the control of innate immune response in cancer.

Ligands for receptors of natural killer (NK) cells and CD8(+) cytotoxic T lymphocytes (CTL), such as the inhibitory nonclassical HLA-G, the activating stress-induced major histocompatibility complex class I-related antigens MICA and MICB, and/or the UL16-binding proteins (ULBPs), are often aberrantly expressed upon viral infection and neoplastic transformation, thereby preventing virus-infected or malignant-transformed cells from elimination by immune effector cells. Recently, it has been shown that ligands of both NK and CD8(+) T cells are regulated by a number of cellular and/or viral microRNAs (miRs). These miRs are involved in shaping the antiviral and/or antitumoral immune responses as well as neoplastic growth properties. This review summarizes the expression pattern and function of miRs directed against selected NK and T cell receptor ligands, their putative role in shaping immune surveillance and tumorigenicity, and their clinical relevance. In addition, the potential role of RNA-binding proteins in the post-transcriptional gene regulation of these ligands will be discussed.

Regulatory CD4+ T cells modulate the interaction between NK cells and hepatic stellate cells by acting on either cell type

NK cells regulate liver fibrosis by killing activated hepatic stellate cells (HSCs) and are controlled themselves by immune cells and/or soluble factors. Here, we analysed if CD4(+) regulatory T cells (Tregs) modify the interaction between NK cells and HSCs. The modification of NK cell activity against HSCs was studied in CD56(high)CD16(-) NK cells, using a flow cytometric CD107a degranulation assay and co-cultures with Tregs from healthy donors and patients with hepatitis C, respectively. We studied the underlying mechanisms in detail, applying Treg supernatants, Treg pretreated HSCs, and recombinant IL-8, TGF-ß1, and IL-10 as well as blocking experiments with neutralizing antibodies and analysed Treg-associated changes in the expression of NK cell receptor ligands on HSCs. Tregs suppressed NK cell activation during HSC co-culture in a cell-contact-dependent manner involving the cytotoxic T-lymphocyte antigen 4 (CTLA-4). NK cell degranulation was further reduced, when HSCs had been pretreated with Tregs (p=0.043), Treg supernatants (p=0.001) or recombinant IL-8 (R=0.630, p=0.001) and TGF-ß1 (R=0.608, p=0.002), respectively. This additional inhibitory effect corresponded to the IL-8/TGF-ß1-mediated downregulation of MIC-A/B and HLA class-I on HSCs. Tregs from hepatitis C likewise inhibited NK cell activity, which was reversed significantly in specific blocking experiments. Our data indicate that Tregs interfere with NK cell regulation of fibrogenesis via both direct cell-contact-dependent inhibition of NK cells and release of soluble factors, downregulating activating NK cell receptor ligands on HSCs. Our data may be particularly relevant for the intrahepatic accumulation of Tregs in chronic hepatitis C because downregulated NK cell activity against HSCs may blunt their control of fibrogenesis.

Examination of galectin-induced lattice formation on early B-cell development.

Galectin-1 (GAL1) is a pre-B cell receptor (pre-BCR) ligand that induces pre-BCR clustering and leads to efficient pre-B cell proliferation and differentiation in the bone marrow. To study pre-BCR-GAL1 interactions and its functional consequence on the early steps of the B cell development, we combine structural nuclear magnetic resonance (NMR) approaches and B cell biology techniques. NMR is applied to identify the residues involved in pre-BCR-GAL1 interactions by monitoring chemical shift perturbations when the complex is formed. This structural information is then used at the cellular level to target specifically the complex formation during GAL1-induced pre-BCR clustering and lattice formation, using immunofluorescence techniques. Moreover, an in vivo assay was set up to study the consequence of synapse formation on the early steps of B cell development.

Poly(ethylene oxide) brushes prepared by the “grafting to” method as a platform for the assessment of cell receptor–ligand binding

Poly(ethylene oxide) (PEO) with terminal alkyne and amino groups was grafted to a poly(glycidyl methacrylate) (PGMA) anchoring layer and the PEO/PGMA coatings were investigated as a non-fouling platform for the assessment of ligand–cell receptor interactions. The PEO/PGMA coatings deposited on Si/SiO2 substrate were stable in phosphate buffered saline over a period of 8days if the thickness of the PEO was less than 30nm. The stability of the coating could be enhanced by an additional layer of 3-mercaptopropyltrimethoxysilane between the substrate and the PGMA layer. The grafted layers were shown to efficiently suppress nonspecific protein adsorption and cell adhesion. Based on the theoretical ligand-binding capacity, protein adsorption and stability data, the optimum thickness range of the PEO layer is 10–20nm. The binding of an arginine–glycine–aspartic acid (RGD) ligand using azide–alkyne click chemistry demonstrated that the ligand surface density is controllable in the range from 100pmol/cm2 up to the capacity of the grafted layer of 102pmol/cm2 by varying the ligand concentration in the reaction mixture. Calf pulmonary artery endothelial cells adhered to and spread on ligand modified layers proportionally to the ligand surface density, thus demonstrating the applicability of these “grafted to” PEO brushes as a platform for cell receptor–ligand engagement studies.

A Stay of Execution: Type I Interferons Pardon T Cells from Death by Natural Killers

In this issue of Immunity, Crouse etal., (2014) and Xu etal., (2014), show that by modulating the expression of natural killer (NK) cell receptor ligands, type I interferons protect responding Tcells against culling by NK cells.

Lectin-like transcript-1 (LLT1) and exosomal PCNA inhibit NK cell effector function against prostate cancer cells (TUM2P.892)

Abstract NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. In order to establish and grow, cancer cells must escape NK cell-mediated killing. By altering the expression of ligands for NK cell receptors (down-regulating activating ligands and up-regulating inhibitory ligands) cancer cells may evade NK mediated killing. Here we have examined the expression of LLT1 (Lectin-like transcript-1 or CLEC2D), a ligand for the NK cell inhibitory receptor NKRP1A (CD161) as well as proliferating cell nuclear antigen (PCNA), a ligand for NKp44 on surface of prostate cancer cell DU145. Expression of LLT1 on DU145 cell inhibited NK cell cytolytic activity though NKRP1A (CD161). Similarly, expression of exosomal PCNA by DU145 inhibited NK cell cytotoxicity through NKp44. The association of HLA I and PCNA on DU145 forms an inhibitory ligand for NKp44. Blocking the inhibitory signals by anti-LLT1 and anti-NKp44 monoclonal antibodies enhanced the killing of prostate cancer cells by NK cells. Thus tumor cells may use multiple pathways to inhibit NK effector function and combinatorial targeting of these pathways could lead to efficient elimination of cancer cells.

Focusing in on T Cell Cross-Reactivity

To provide broad immunity to a vast array of foreign antigens with a limited number of T lymphocytes, each cell has to recognize many targets. By implementing a strategy to identify T cell receptor (TCR) ligands and investigating at a fine granularity their structure and sequence relationship, Birnbaum et al. demonstrate the surprisingly tight focus of such T cell cross-reactivity.

KIR2DL3+NKG2A− natural killer cells are associated with protection from productive hepatitis C virus infection in people who inject drugs

Despite continuous high-risk behavior, a subgroup among people who inject drugs (PWID) remains seronegative for hepatitis C virus (HCV) suggesting that a state of "natural resistance" to HCV Infection may exist. Homozygosity for KIR2DL3 and its ligand HLA-C1 group alleles has been associated with control of HCV infection, however, the mechanism mediating this protective effect remained unclear. Peripheral NK cells from PWID (n=104) were phenotypically and functionally characterized by multicolor flow cytometry. Expression levels of the NK cell receptor ligands were analysed in liver biopsies and primary human hepatocytes. HCV seronegative PWID (n=34) had increased levels of KIR2DL3(+)NKG2A(-) NK cells compared to healthy controls (n=10; p<0.001) and PWID with chronic (n=38; p<0.001) or resolved infection (n=37; p<0.001). There was an inverse correlation between the frequency of KIR2DL3(+) and NKG2A(+) NK cells (r=-0.53; p<0.0001). Importantly, expression of HLA-E, the ligand for NKG2A, was significantly upregulated in liver biopsies of HCV infected patients (n=51) compared to HBV infected patients (n=22; p<0.01) and correlated with HCV viral load (r=0.32; p<0.0029). In functional analyses KIR2DL3(-)NKG2A(+) NK cells but not KIR2DL3(+)NKG2A(-) NK cells were significantly inhibited by HLA-E ligation. Accordingly, interferon gamma secretion of NK cells from PWID with chronic infection but not from HCV seronegative PWID was significantly suppressed in the presence of HLA-E. KIR2DL3(+)NKG2A(-) NK cells are not sensitive to HLA-E-mediated inhibition and may thereby control early HCV infection prior to seroconversion and result in an apparent state of "natural resistance" to HCV in PWID.

Tissue-Specific Microvascular Endothelial Cells Show Distinct Capacity To Activate NK Cells: Implications for the Pathophysiology of Granulomatosis with Polyangiitis

The relevance of tissue specificity of microvascular endothelial cells (MECs) in the response to inflammatory stimuli and sensitivity to immune cell-mediated injury is not well defined. We hypothesized that such MEC characteristics might shape their interaction with NK cells through the use of different adhesion molecules and NK cell receptor ligands or the release of different soluble factors and render them more or less vulnerable to NK cell injury during autoimmune vasculitis, such as granulomatosis with polyangiitis (GPA). To generate a comprehensive expression profile of human MECs of renal, lung, and dermal tissue origin, we characterized, in detail, their response to inflammatory cytokines and to proteinase 3, a major autoantigen in GPA, and analyzed the effects on NK cell activation. In this study, we show that renal MECs were more susceptible than lung and dermal MECs to the effect of inflammatory signals, showing upregulation of ICAM-1 and VCAM-1 on their surface, as well as release of CCL2, soluble fractalkine, and soluble VCAM-1. Proteinase 3-stimulated renal and lung MECs triggered CD107a degranulation in control NK cell. Notably, NK cells from GPA patients expressed markers of recent in vivo activation (CD69, CD107a), degranulated more efficiently than did control NK cells in the presence of renal MECs, and induced direct killing of renal MECs in vitro. These results suggest that, upon inflammatory conditions in GPA, renal MECs may contribute to the recruitment and activation of NK cells in the target vessel wall, which may participate in the necrotizing vasculitis of the kidney during this disease.

Quantifying the Protection of Activating and Inhibiting NK Cell Receptors during Infection with a CMV-Like Virus

The responsiveness of natural killer (NK) cells is controlled by balancing signals from activating and inhibitory receptors. The most important ligands of inhibitory NK cell receptors are the highly polymorphic major histocompatibility complex (MHC) class I molecules, which allow NK cells to screen the cellular health of target cells. Although these inhibitory receptor–ligand interactions have been well characterized, the ligands for most activating receptors are still unknown. The mouse cytomegalovirus (MCMV) represents a helpful model to study NK cell-driven immune responses. Many studies have demonstrated that CMV infection can be controlled by NK cells via their activating receptors, but the exact contribution of the different signaling potential (i.e., activating vs. inhibiting) remains puzzling. In this study, we have developed a probabilistic model, which predicts the optimal specificity of inhibitory and activating NK cell receptors needed to offer the best protection against a CMV-like virus. We confirm our analytical predictions with an agent-based model of an evolving host population. Our analysis quantifies the degree of protection of each receptor type, revealing that mixed haplotypes (i.e., haplotypes composed of activating and inhibiting receptors) are most protective against CMV-like viruses, and that the protective effect depends on the number of MHC loci per individual.

NK Cell Mediated Cytotoxicity Against Malignant Promyelocytes Enhanced By Arsenic Trioxide: Potential Clinical Relevance

Abstract NK cells are primary effectors of the innate immune response against cells that have undergone malignant transformation. Several lines of evidence indicate that the expression level of NK ligands on leukemic cells affects the sensitivity of the leukemic cells to cytolytic activity by NK cells. Various agents have been evaluated for their ability to induce these ligands on leukemic cells to augment the NK cell mediated anti-leukemia effect. There is substantial evidence that has established the importance of the adaptive immune system in the treatment of acute promyelocytic leukemia (APL) (Rose Ann Padua et al. Nat Med 2003). While there is significant data which address the mechanisms of arsenic trioxide (ATO) on malignant promyelocytes, limited data is available of its effect on the innate and adaptive immune system. We undertook a series of experiments to address the impact of ATO on NK cell receptor and malignant promyelocyte ligand expression and its effect on NK cell mediated cytotoxicity. We also evaluated NK cell reconstitution in patients treated with ATO and the impact of KIR genotypes on relapse. We first evaluated the cytotoxic activity of NK92MI (NK cell line) against 5 different myeloid (K562, U937, HL60, UF1, NB4) and 2 lymphoid cell lines (Jurkat E6.1, SUP-B15) by CFSE/ 7AAD cytotoxicity assay. Target (T) cells (1x 105/100 µL/well) pre-treated with CFSE were co-cultured with effector NK cells (E) at a E:T ratio of 1:1, 2:1 and 5:1 for 5 hours at 37°C in 96 well plates. The percentage cytolytic activity of the NK cells was then calculated after adding 7AAD and acquired in FACS Calibur (Becton Dickinson, San Jose, CA, USA). Significant cytolytic activity was noted against K562 and NB4 cell lines. At the highest E:T ratio there was a median 22% cytolytic activity against NB4 (N=5). We observed that NB4 when treated overnight with 1µM ATO (&gt;99% viability retained after this exposure) significantly increased the cytotoxic effect of NK92MI cell line at all the E:T ratios as shown in figure 1A (n=5; P=0.0023). No other cell line showed a similar increase in cytotoxic effect following exposure to ATO at these concentrations (data not shown). We next evaluated the effect of exposure of NB4 cells to ATO at 1µM for 6 hours on NK ligand expression by flowcytometry. As shown in figure 1B there was a significant increase in activating ligand MICA/B in NB4 cell lines (n=3; P=0.016) which was not seen in any of the other cell lines. Similar significant increased expression of Nectin-2 (DNAM-1 ligand) and HLA Class I was seen. Exposure of NK92MI to ATO for 6 hours at 1uM (non cytotoxic dose:IC50-3.8uM) resulted in increased expression of activating receptors NKG2D, NKP30 and KIR2DS4 (figure 1C) and inhibitory receptor NKG2A and decrease in inhibitory receptors KIR3DL1/DL2. There were no changes in the expression of NKP46, KIR2DL1, KIR2DL2 and DNAM1 receptors. We undertook a prospective study to evaluate the pattern of NK (CD56+CD3-) reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen (Mathews et al. JCO 2011). The mean NK cell counts in patients were below the 2SD deviation level of the normal range even after completion of therapy (approximately a year)(figure 1D). All other subsets evaluated (CD4, CD8, CD3, CD19, CD56+CD3+, CD4CD45RO) had returned to levels within the normal range by the end of consolidation therapy (approximately 3 months from diagnosis). KIR genotyping was done on 55 patients with APL who received treatment with single agent ATO based regimen. The median follow up of this cohort was 20 months and 14 cases relapsed following initial therapy. The presence or absence of 17 KIR genes was done by PCR-SSP method (KIR Typing kit, Miltenyi Biotech Inc, CA). There was no association with any specific genotype or haplotype with risk of relapse. In summary we have noted that there is up regulation of receptors on NK cells and ligands on malignant promyelocytes following exposure to ATO that favors NK cell mediated cytotoxicity. In-vitro we have demonstrated a significant increase in NK cell mediated cytolytic activity against malignant promyelocytes exposed to ATO even at relatively low E:T ratios. This could be an important mechanism by which ATO induces durable remissions in patients with APL. The delayed NK cell recovery following treatment with ATO raises the possibility of using NK cell therapy to augment the effect of ATO in the treatment of patients.Figure 1Figure 1. Disclosures: No relevant conflicts of interest to declare.

Innate immune control of EBV-infected B cells by invariant natural killer T cells

AbstractIndividuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon γ secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition.

Low-Affinity T Cells Are Programmed to Maintain Normal Primary Responses but Are Impaired in Their Recall to Low-Affinity Ligands

T cell responses to low-affinity T cell receptor (TCR) ligands occur in the context of infection, tumors, and autoimmunity despite diminished TCR signal strength. The processes that enable such responses remain unclear. We show that distinct mechanisms drive effector/memory development in high- and low-affinity T cells. Low-affinity cells preferentially differentiate into memory precursors of a central memory phenotype that are interleukin (IL)-12R(lo), IL-7R(hi), and Eomes(hi). Strikingly, in contrast to naive cells, low-affinity memory cells were impaired in the response to low- but not high-affinity ligands, indicating that low-affinity cells are programmed to generate diverse immune responses while avoiding autoreactivity. Affinity and antigen dose directly correlated with IL-12R signal input and T-bet but not with Eomes expression because low- affinity signals were more potent inducers of Eomes at a high antigen dose. Our studies explain how weak antigenic signals induce complete primary immune responses and provide a framework for therapeutic intervention.

FRI0020 Blocking the inhibitory CD94/NKG2A NK cell receptor with a novel ANTI-NKG2A MAB enhances the susceptibility of rheumatoid arthritis fibroblast-like synoviocytes (FLS) to NK cell-mediated cytotoxicity

BackgroundRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive bone erosion and cartilage degradation [1]. Fibroblast-like synoviocytes (FLS) present in the inflamed...

Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity

AbstractNatural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

Partial MHC class II constructs inhibit MIF/CD74 binding and downstream effects (P5221)

Abstract Macrophage migration inhibitory factor (MIF) and its receptor, CD74, are pivotal regulators of the immune system. Here we demonstrate for the first time that partial MHC class II constructs comprised of linked beta 1 alpha 1 domains with covalently attached antigenic peptides (also referred to as recombinant T cell receptor ligands - RTL) can inhibit MIF activity by not only blocking the binding of rhMIF to immunopurified CD74, but also down-regulating CD74 cell-surface expression. This bi-functional inhibition of MIF/CD74 interactions blocks downstream MIF effects, including enhanced secretion of proinflammatory cytokines, anti-apoptotic activity and inhibition of random migration that all contribute to reversal of clinical and histological signs of experimental autoimmune encephalomyelitis (EAE). Moreover, we demonstrate that enhanced CD74 cell surface expression on monocytes in mice with EAE and subjects with multiple sclerosis (MS) can be down-regulated by humanized RTL, resulting in reduced MIF binding to the cells. Thus, binding of partial MHC complexes to CD74 blocks both the accessibility and availability of CD74 for MIF binding and downstream inflammatory activity.

In vivo performance of polymer nanocarriers dually-targeted to epitopes of the same or different receptors

Modification of drug delivery nanomaterials with affinity molecules that facilitate targeting, has rendered a new class of ligands for cell receptors, which often possess valency and dimensions different from natural counterparts. Designing strategies to target multiple receptors or, never explored, multiple epitopes on the same receptor may modulate the biodistribution properties of these nanomaterials. We examined this using antibody-directed targeting of polymer nanocarriers to transferrin receptor (TfR) and intercellular adhesion molecule 1 (ICAM-1). Regarding epitopes on one receptor, nanocarriers addressed with anti-TfR-R17 maintained brain and lung targeting in mice, compared with “free” antibody, while anti-TfR-8D3 nanocarriers lost specificity. Coating nanocarriers with both antibodies decreased targeting in brain and liver, not lungs, modulating biodistribution. Regarding different receptors, nanocarriers coated with both anti-ICAM and anti-TfR displayed intermediate specific accumulation in lungs and higher in liver, compared to single-targeted nanocarriers, while brain targeting was comparable to TfR- and lower than ICAM-1-targeted nanocarriers. Tracing a model therapeutic cargo, acid sphingomyelinase (enzyme replacement for Niemann–Pick Disease A-B), showed that combined-targeted anti-ICAM/TfR nanocarriers enhanced enzyme delivery versus “free” enzyme, with biodistribution patterns different from single-targeted nanocarriers. Hence, targeting nanocarriers to multiple epitopes or receptors holds promise to control distribution of drug delivery nanomaterials in the body.

Effective Arrestin–Specific Immunotherapy of Experimental Autoimmune Uveitis with RTL: A Prospect for Treatment of Human Uveitis

To evaluate the immunotherapeutic efficacy of recombinant T cell receptor ligands (RTLs) specific for arrestin immunity in treatment of experimental autoimmune uveitis (EAU) in humanized leukocyte antigen (HLA-DR3) transgenic (Tg) mice. We generated de novo recombinant human DR3-derived RTLs bearing covalently tethered arrestin peptides 291-310 (RTL351) or 305-324 (RTL352). EAU was induced by immunization of HLA-DR3 mice with arrestin or arrestin peptide and treated with RTLs by subcutaneous delivery. T cell proliferation and cytokine expression was measured in RTL-treated and control mice. RTL351 prevented the migration of cells outside of the spleen and the recruitment of inflammatory cells into the eye, and provided full protection against inflammation from EAU induced with arrestin or arrestin peptides. RTL351 significantly inhibited T cell proliferation and secretion of inflammatory cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), IL-6, and IL-17 and chemokines (macrophage inflammatory proteins [MIP-1a] and regulated and normal T cell expressed and secreted [RANTES]), which is in agreement with the suppression of intraocular inflammation. RTL350 ("empty," no peptide) and RTL352 were not effective. Immunotherapy with a single RTL351 successfully prevented and treated arrestin-induced EAU in HLA-DR3 mice and provided proof of concept for therapy of autoimmune uveitis in human patients. The beneficial effects of RTL351 should be attributed to a significant decrease in Th1/Th17 mediated inflammation. Successful therapies for autoimmune uveitis must specifically inhibit pathogenic inflammation without inducing generalized immunosuppression. RTLs can offer such an option. The single retina-specific RTLs may have a value as potential immunotherapeutic drug for human autoimmune uveitis because they effectively prevent disease induced by multiple T cell specificities.