Artificial Antigen Presenting Cells for Detection and Desensitization of Autoreactive T cells Associated with Type 1 Diabetes.

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen-presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC, we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients, and using mouse models, we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.

The Intracellular Proteome as a Source for Novel Targets in CAR-T and T-Cell Engagers-Based Immunotherapy.

The impressive clinical success of cancer immunotherapy has motivated the continued search for new targets that may serve to guide potent effector functions in an attempt to efficiently kill malignant cells. The intracellular proteome is an interesting source for such new targets, such as neo-antigens and others, with growing interest in their application for cell-based immunotherapies. These intracellular-derived targets are peptides presented by MHC class I molecules on the cell surface of malignant cells. These disease-specific class I HLA-peptide complexes can be targeted by specific TCRs or by antibodies that mimic TCR-specificity, termed TCR-like (TCRL) antibodies. Adoptive cell transfer of TCR engineered T cells and T-cell-receptor-like based CAR-T cells, targeted against a peptide-MHC of interest, are currently tested as cancer therapeutic agents in pre-clinical and clinical trials, along with soluble TCR- and TCRL-based agents, such as immunotoxins and bi-specific T cell engagers. Targeting the intracellular proteome using TCRL- and TCR-based molecules shows promising results in cancer immunotherapy, as exemplified by the success of the anti-gp100/HLA-A2 TCR-based T cell engager, recently approved by the FDA for the treatment of unresectable or metastatic uveal melanoma. This review is focused on the selection and isolation processes of TCR- and TCRL-based targeting moieties, with a spotlight on pre-clinical and clinical studies, examining peptide-MHC targeting agents in cancer immunotherapy.

Serologic Status and Toxic Effects of the SARS-CoV-2 BNT162b2 Vaccine in Patients Undergoing Treatment for Cancer.

Importance: The efficacy and safety profile of SARS-CoV-2 vaccines have been acquired from phase 3 studies; however, patients with cancer were not represented in these trials. Owing to the recommendation to prioritize high-risk populations for vaccination, further data are warranted. Objective: To evaluate the use and safety of the BNT162b2 vaccine in patients undergoing treatment for cancer. Design, Setting, and Participants: In January 2021, mass SARS-CoV-2 vaccination of high-risk populations, including patients with cancer, was initiated in Israel. This cohort study prospectively enrolled and followed up patients with cancer and healthy participants between January 15 and March 14, 2021. The study was conducted at the Division of Oncology of Rambam Health Care Campus, the major tertiary (referral) medical center of northern Israel. Participants included 232 patients with cancer who were receiving active treatment after the first and second doses of the BNT162b2 vaccine and 261 healthy, age-matched health care workers who served as controls. Exposures: Serum samples were collected after each vaccine dose and in cases of seronegativity. Questionnaires regarding sociodemographic characteristics and adverse reactions were administered at serum collection. A regulatory agencies-approved assay was used to assess IgG at all time points. Patients' electronic medical records were reviewed for documentation of COVID-19 infection and results of blood cell counts, liver enzyme levels, and imaging studies. Main Outcomes and Measures: Seroconversion rate after the first and second doses of the BNT162b2 vaccine and documented COVID-19 infection. Results: Of the 232 patients undergoing treatment for cancer, 132 were men (57%); mean (SD) age was 66 (12.09) years. After the first dose of BNT162b2 vaccine, 29% (n = 25) patients were seropositive compared with 84% (n = 220) of the controls (P < .001). After the second dose, the seropositive rate reached 86% (n = 187) in the patients. Testing rate ratios per 1000 person-days after the first dose were 12.5 (95% CI, 3.4-45.7) for the patients and 48.5 (95% CI, 37.2-63.2) for the controls. Patients undergoing chemotherapy showed reduced immunogenicity (odds ratio, 0.41; 95% CI, 0.17-0.98). In seronegative patients, the rate of documented absolute leukopenia reached 39%. No COVID-19 cases were documented throughout the study period; however, 2 cases in the patient cohort were noted immediately after the first dose. Reported adverse events were similar to data in former trials comprising mostly healthy individuals. Conclusions and Relevance: In this cohort study, the SARS-CoV-2 BNT162b2 vaccine appeared to be safe and achieve satisfactory serologic status in patients with cancer. There was a pronounced lag in antibody production compared with the rate in noncancer controls; however, seroconversion occurred in most patients after the second dose. Future real-world data are warranted to determine the long-term efficacy of the vaccine with regard to type of anticancer treatment.

Phenotypic Models of CAR T-Cell Activation Elucidate the Pivotal Regulatory Role of CAR Downmodulation.

Adoptive cell immunotherapy with chimeric antigen receptor (CAR) showed limited potency in solid tumors, despite durable remissions for hematopoietic malignancies. Therefore, an investigation of ways to enhance the efficacy of CARs' antitumor response has been engaged upon. We previously examined the interplay between the biophysical parameters of CAR binding (i.e., affinity, avidity, and antigen density), as regulators of CAR T-cell activity and detected nonmonotonic behaviors of affinity and antigen density and an interrelation between avidity and antigen density. Here, we built an evolving phenotypic model of CAR T-cell regulation, which suggested that receptor downmodulation is a key determinant of CAR T-cell function. We verified this assumption by measuring and manipulating receptor downmodulation and intracellular signaling processes. CAR downmodulation inhibition, via actin polymerization inhibition, but not inhibition of regulatory inhibitory phosphatases, was able to increase CAR T-cell responses. In addition, we documented trogocytosis in CAR T cells that depends on actin polymerization. In summary, our study modeled the parameters that govern CAR T-cell engagement and revealed an underappreciated mechanism of T-cell regulation. These results have a potential to predict and therefore advance the rational design of CAR T cells for adoptive cell treatments.See related article on p. 872.

Shaping Functional Avidity of CAR T Cells: Affinity, Avidity, and Antigen Density That Regulate Response.

Chimeric antigen receptors (CARs) are immunoreceptors that redirect T cells to selectively kill tumor cells. Given their clinical successes in hematologic malignancies, there is a strong aspiration to advance this immunotherapy for solid cancers; hence, molecular CAR design and careful target choice are crucial for their function. To evaluate the functional significance of the biophysical properties of CAR binding (i.e., affinity, avidity, and antigen density), we generated an experimental system in which these properties are controllable. We constructed and characterized a series of CARs, which target the melanoma tumor-associated antigen Tyr/HLA-A2, and in which the affinity of the single-chain Fv binding domains ranged in KD from 4 to 400 nmol/L. These CARs were transduced into T cells, and each CAR T-cell population was sorted by the level of receptor expression. Finally, the various CAR T cells were encountered with target cells that present different levels of the target antigen. We detected nonmonotonic behaviors of affinity and antigen density, and an interrelation between avidity and antigen density. Antitumor activity measurements in vitro and in vivo corroborated these observations. Our study contributes to the understanding of CAR T-cell function and regulation, having the potential to improve therapies by the rational design of CAR T cells.See related article on p. 946.

Multi-center nationwide comparison of seven serology assays reveals a SARS-CoV-2 non-responding seronegative subpopulation.

BACKGROUND: An Israeli national taskforce performed a multi-center clinical and analytical validation of seven serology assays to determine their utility and limitations for SARS-CoV-2 diagnosis. METHODS: Serology assays from Roche, Abbott, Diasorin, BioMerieux, Beckman-Coulter, Siemens, and an in-house RBD ELISA were included. Negative samples from 2391 individuals representative of the Israeli population, and 698 SARS-CoV-2 PCR positive patients, collected between March and May 2020, were analyzed. FINDINGS: Immunoassays sensitivities between 81.5%-89.4% and specificities between 97.7%-100% resulted in a profound impact on the expected Positive Predictive Value (PPV) in low (<15%) prevalence scenarios. No meaningful increase was detected in the false positive rate in children compared to adults. A positive correlation between disease severity and antibody titers, and no decrease in antibody titers in the first 8 weeks after PCR positivity was observed. We identified a subgroup of symptomatic SARS-CoV-2 positive patients (~5% of patients), who remained seronegative across a wide range of antigens, isotypes, and technologies. INTERPRETATION: The commercially available automated immunoassays exhibit significant differences in performance and expected PPV in low prevalence scenarios. The low false-positivity rate in under 20's suggests that cross-reactive immunity from previous CoV strains is unlikely to explain the milder disease course in children. Finding no decrease in antibody titers in the first 8 weeks is in contrast to some reports of short half-life for SARS-CoV-2 antibodies. The ~5% who were seronegative non-responders, using multiple assays in a population-wide manner, represents the proportion of patients that may be at risk for re-infection. FUNDING: Israel Ministry of Health.

SS1P Immunotoxin Induces Markers of Immunogenic Cell Death and Enhances the Effect of the CTLA-4 Blockade in AE17M Mouse Mesothelioma Tumors.

SS1P is an anti-mesothelin immunotoxin composed of a targeting antibody fragment genetically fused to a truncated fragment of Pseudomonas exotoxin A. Delayed responses reported in mesothelioma patients receiving SS1P suggest that anti-tumor immunity is induced. The goal of this study is to evaluate if SS1P therapy renders mesothelioma tumors more sensitive to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) immune checkpoint blockade. We evaluated the ability of SS1P to induce adenosine triphosphate (ATP) secretion and calreticulin expression on the surface of AE17M mouse mesothelioma cells. Both properties are associated with immunogenic cell death. Furthermore, we treated these tumors with intra-tumoral SS1P and systemic CTLA-4. We found that SS1P increased the release of ATP from AE17M cells in a dose and time-dependent manner. In addition, SS1P induced calreticulin expression on the surface of AE17M cells. These results suggest that SS1P promotes immunogenic cell death and could sensitize tumors to anti-CTLA-4 based therapy. In mouse studies, we found that the combination of anti-CTLA-4 with intra-tumoral SS1P induced complete regressions in most mice and provided a statistically significant survival benefit compared to monotherapy. The surviving mice were protected from tumor re-challenge, indicating the development of anti-tumor immunity. These findings support the use of intra-tumoral SS1P in combination with anti-CTLA-4.

Combining Local Immunotoxins Targeting Mesothelin with CTLA-4 Blockade Synergistically Eradicates Murine Cancer by Promoting Anticancer Immunity.

Immune checkpoint blockade using antibodies to cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) benefits a limited number of cancer patients. SS1P and LMB-100 are immunotoxins that target mesothelin. We observed delayed responses to SS1P in patients with mesothelioma suggesting that antitumor immunity was induced. Our goal was to stimulate antitumor immunity by combining SS1P or LMB-100 with anti-CTLA-4. We constructed a BALB/c breast cancer cell line expressing human mesothelin (66C14-M), which was implanted in one or two locations. SS1P or LMB-100 was injected directly into established tumors and anti-CTLA-4 administered i.p. In mice with two tumors, one tumor was injected with immunotoxin and the other was not. The complete regression rate was 86% for the injected tumors and 53% for the uninjetced tumors. No complete regressions occurred when drugs were given separately. In regressing tumors, dying and dead tumor cells were intermingled with PMNs and surrounded by a collar of admixed eosinophils and mononuclear cells. Tumor regression was associated with increased numbers of tumor-infiltrating CD8+ cells and blocked by administration of antibodies to CD8. Surviving mice were protected from tumor rechallenge by 66C14 cells not expressing mesothelin, indicating the development of antitumor immunity. The antitumor effect was abolished when a mutant noncytotoxic variant was used instead of LMB-100, showing that the antitumor response is not mediated by recognition of a foreign bacterial protein. Our findings support developing a therapy composed of immunotoxins and checkpoint inhibitors for patients. Cancer Immunol Res; 5(8); 685-94. ©2017 AACR.

Major-histocompatibility complex harnessing and lymphocytic recruitment: a new tool for glioblastoma multiforme immunotherapy.

Glioblastoma multiforme (GBM) is the most common malignant primary brain neoplasm, notorious for being able to evade and suppress the immune-system. We present a new immunotherapeutic approach that can potentially overcome or circumvent many of the GBM laden obstacles for an efficient antitumor immune response. In this molecular construct, a soluble major-histocompatibility complex (MHC), presenting an immunogenic peptide is directed towards GBM cells. This soluble complex is known to be able to activate relevant cytotoxic T-lymphocyte (CTL's) populations, mounting an effective local immune response. The chimeric protein consists of a targeting domain, a function that can be fulfilled by an antibody or other small molecule that binds tumor associated antigens, and an effector domain. A single chain MHC directly linked to an immunogenic Cytomegalovirus derived peptide (phosphoprotein 65) can be used as such an effector domain. Targeting MHC complexes to the tumor enables to recruit different lymphocyte populations using MHC-molecules bearing a single, highly antigenic peptide derived from immunogenic T cell epitopes. Moreover, the recruited potent memory CTL's at the tumor's milieu may prove resistant to the previously described local immunosuppressive environment, and may enable the shift to TH1 cytokine profile resulting in specific massive tumor destruction.

Recruitment of Oligoclonal Viral-Specific T cells to Kill Human Tumor Cells Using Single-Chain Antibody-Peptide-HLA Fusion Molecules.

Tumor progression is often associated with the development of diverse immune escape mechanisms. One of the main tumor escape mechanism is HLA loss, in which human solid tumors exhibit alterations in HLA expression. Moreover, tumors that present immunogenic peptides via class I MHC molecules are not susceptible to CTL-mediated lysis, because of the relatively low potency of the tumor-specific CLTs. Here, we present a novel cancer immunotherapy approach that overcomes these problems by using the high affinity and specificity of antitumor antibodies to recruit potent antiviral memory CTLs to attack tumor cells. We constructed a recombinant molecule by genetic fusion of a cytomegalovirus (CMV)-derived peptide pp65 (NLVPMVATV) to scHLA-A2 molecules that were genetically fused to a single-chain Fv Ab fragment specific for the tumor cell surface antigen mesothelin. This fully covalent fusion molecule was expressed in E. coli as inclusion bodies and refolded in vitro. The fusion molecules could specifically bind mesothelin-expressing cells and mediate their lysis by NLVPMVATV-specific HLA-A2-restricted human CTLs. More importantly, these molecules exhibited very potent antitumor activity in vivo in a nude mouse model bearing preestablished human tumor xenografts that were adoptively transferred along with human memory CTLs. These results represent a novel and powerful approach to immunotherapy for solid tumors, as demonstrated by the ability of the CMV-scHLA-A2-SS1(scFv) fusion molecule to mediate specific and efficient recruitment of CMV-specific CTLs to kill tumor cells.

A novel host-proteome signature for distinguishing between acute bacterial and viral infections.

Bacterial and viral infections are often clinically indistinguishable, leading to inappropriate patient management and antibiotic misuse. Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogens. Our aim was to identify novel viral-induced host proteins that can complement bacterial-induced proteins to increase diagnostic accuracy. Initially, we conducted a bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria. The best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. We further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96). The signature was superior to any of the individual proteins (P<0.001), as well as routinely used clinical parameters and their combinations (P<0.001). It remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse.

Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody-based chimeric antigen receptors indicates affinity/avidity thresholds.

Adoptive transfer of Ag-specific T lymphocytes is an attractive form of immunotherapy for cancers. However, acquiring sufficient numbers of host-derived tumor-specific T lymphocytes by selection and expansion is challenging, as these cells may be rare or anergic. Using engineered T cells can overcome this difficulty. Such engineered cells can be generated using a chimeric Ag receptor based on common formats composed from Ag-recognition elements such as αß-TCR genes with the desired specificity, or Ab variable domain fragments fused with T cell-signaling moieties. Combining these recognition elements are Abs that recognize peptide-MHC. Such TCR-like Abs mimic the fine specificity of TCRs and exhibit both the binding properties and kinetics of high-affinity Abs. In this study, we compared the functional properties of engineered T cells expressing a native low affinity αß-TCR chains or high affinity TCR-like Ab-based CAR targeting the same specificity. We isolated high-affinity TCR-like Abs recognizing HLA-A2-WT1Db126 complexes and constructed CAR that was transduced into T cells. Comparative analysis revealed major differences in function and specificity of such CAR-T cells or native TCR toward the same antigenic complex. Whereas the native low-affinity αß-TCR maintained potent cytotoxic activity and specificity, the high-affinity TCR-like Ab CAR exhibited reduced activity and loss of specificity. These results suggest an upper affinity threshold for TCR-based recognition to mediate effective functional outcomes of engineered T cells. The rational design of TCRs and TCR-based constructs may need to be optimized up to a given affinity threshold to achieve optimal T cell function.

HLA-DRα1 constructs block CD74 expression and MIF effects in experimental autoimmune encephalomyelitis.

CD74, the cell-surface form of the MHC class II invariant chain, is a key inflammatory factor that is involved in various immune-mediated diseases as part of the macrophage migration inhibitory factor (MIF) binding complex. However, little is known about the natural regulators of CD74 in this context. In order to study the role of the HLA-DR molecule in regulating CD74, we used the HLA-DRα1 domain, which was shown to bind to and downregulate CD74 on CD11b(+) monocytes. We found that DRα1 directly inhibited binding of MIF to CD74 and blocked its downstream inflammatory effects in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). Potency of the DRα1 domain could be destroyed by trypsin digestion but enhanced by addition of a peptide extension (myelin oligodendrocyte glycoprotein [MOG]-35-55 peptide) that provided secondary structure not present in DRα1. These data suggest a conformationally sensitive determinant on DRα1-MOG that is responsible for optimal binding to CD74 and antagonism of MIF effects, resulting in reduced axonal damage and reversal of ongoing clinical and histological signs of EAE. These results demonstrate natural antagonist activity of DRα1 for MIF that was strongly potentiated by the MOG peptide extension, resulting in a novel therapeutic, DRα1-MOG-35-55, that within the limitations of the EAE model may have the potential to treat autoimmune diseases such as multiple sclerosis.

On the limited recognition of inorganic surfaces by short peptides compared with antibodies.

The vast potential applications of biomolecules that bind inorganic surfaces led mostly to the isolation of short peptides that target selectively specific materials. The demonstrated differential affinity toward certain surfaces created the impression that the recognition capacity of short peptides may match that of rigid biomolecules. In the following, we challenge this view by comparing the capacity of antibody molecules to discriminate between the (100) and (111A) facets of a gallium arsenide semiconductor crystal with the capacity of short peptides to do the same. Applying selection from several peptide and single chain phage display libraries, we find a number of antibody molecules that bind preferentially a given crystal facet but fail to isolate, in dozens of attempts, a single peptide capable of such recognition. The experiments underscore the importance of rigidity to the recognition of inorganic flat targets and therefore set limitations on potential applications of short peptides in biomimetics.

Antigen-dependent integration of opposing proximal TCR-signaling cascades determines the functional fate of T lymphocytes.

T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses lead to the unresponsive phenotype. How T cells "measure" Ag dose and how this determines functional output to a given antigenic dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation- and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide-MHC complexes/cell was found to be the transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state. Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in the presence of very high Ag doses. This mechanism permits T cells to make integrated "measurements" of Ag dose that determine subsequent functional outcomes.

Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope.

The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic Islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T-cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T-cell epitope GAD-555-567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555-567 complexes in antigen presenting cells. I561M single mutation at the central position (P5) of the GAD-555-567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555-567 specific, DR4 restricted T-cell response in vitro and in vivo in HLA-DR4 transgenic mice. Our findings constitute a proof-of-concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools that enable studies related to antigen presentation as well as novel therapeutic agents that may be used to modulate autoimmune disorders such as T1D.

MUC1 is a potential target for the treatment of acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a malignancy of stem cells with an unlimited capacity for self-renewal. MUC1 is a secreted, oncogenic mucin that is expressed aberrantly in AML blasts, but its potential uses to target AML stem cells have not been explored. Here, we report that MUC1 is highly expressed on AML CD34(+)/lineage(-)/CD38(-) cells as compared with their normal stem cell counterparts. MUC1 expression was not restricted to AML CD34(+) populations as similar results were obtained with leukemic cells from patients with CD34(-) disease. Engraftment of AML stem cell populations that highly express MUC1 (MUC1(high)) led to development of leukemia in NOD-SCID IL2Rgamma(null) (NSG) immunodeficient mice. In contrast, MUC1(low) cell populations established normal hematopoiesis in the NSG model. Functional blockade of the oncogenic MUC1-C subunit with the peptide inhibitor GO-203 depleted established AML in vivo, but did not affect engraftment of normal hematopoietic cells. Our results establish that MUC1 is highly expressed in AML stem cells and they define the MUC1-C subunit as a valid target for their therapeutic eradication.

A novel regulatory pathway for autoimmune disease: binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance.

Treatment with partial (p)MHC class II-ß1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b(+) mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.

Immunoglobulin-like transcript receptors on human dermal CD14+ dendritic cells act as a CD8-antagonist to control cytotoxic T cell priming.

Human Langerhans cells (LCs) are highly efficient at priming cytolytic CD8(+) T cells compared with dermal CD14(+) dendritic cells (DCs). Here we show that dermal CD14(+) DCs instead prime a fraction of naïve CD8(+) T cells into cells sharing the properties of type 2 cytokine-secreting CD8(+) T cells (TC2). Differential expression of the CD8-antagonist receptors on dermal CD14(+) DCs, the Ig-like transcript (ILT) inhibitory receptors, explains the difference between the two types of DCs. Inhibition of CD8 function on LCs inhibited cytotoxic T lymphocytes (CTLs) and enhanced TC2 generation. In addition, blocking ILT2 or ILT4 on dermal CD14(+) DCs enhanced the generation of CTLs and inhibited TC2 cytokine production. Lastly, addition of soluble ILT2 and ILT4 receptors inhibited CTL priming by LCs. Thus, ILT receptor expression explains the polarization of CD8(+) T-cell responses by LCs vs. dermal CD14(+) DCs.

Mixed alkanethiol monolayers on submicrometric gold patterns: a controlled platform for studying cell-ligand interactions.

Nanoscale organization of surface ligands often has a critical effect on cell-surface interactions. We have developed an experimental system that allows a high degree of control over the 2-D spatial distribution of ligands. As a proof of concept, we used the developed system to study how T-cell activation is independently affected by antigen density and antigen amount per cell. Arrays of submicrometer gold islands at varying surface coverage were defined on silicon by electron beam lithography (EBL). The gold islands were functionalized with alkanethiol self-assembled monolayers (SAMs) containing a small antigen, 2,4,6-trinotrophenyl (TNP), at various densities. Genetically engineered T-cell hybridomas expressing TNP-specific chimeric T-cell antigen receptor (CAR) were cultured on the SAMs, and their activation was assessed by IL-2 secretion and CD69 expression. It was found that, at constant antigen density, activation increased monotonically with the amount of antigen, while at constant antigen amount activation was maximal at an intermediate antigen density, whose value was independent of the amount of antigen.