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Antibody-dependent cell-mediated cytotoxicity (ADCC) assay has been widely performed for immuno-oncological research. Traditionally, ADCC assays are conducted by measuring the amount of released Chromium, calcein AM, or Lactate dehydrogenase (LDH) molecules after the target cancer cells are killed by effector/antibody pair. These methods can be inaccurate due to indirect measurement of supernatant at the end point of 4 hour, however, there is a need to characterize the effects of antibody-dependent cytotoxicity for longer than 72 hours. In this work, we demonstrated the time-course monitoring of NK cell-mediated ADCC of A375 cells in the presence or absence of IL2 and antibodies for 76 hours. First, A375-ZsGreen cells were seeded into a 96-well plate with and without target antibodies. Next, NK cells with and without IL2 were added to the wells at 10:1, 5:1, 2:1, and 1:1 E:T ratios. The co-culture was allowed to incubate for 76 hours and the image cytometer was used to scan and analyze at times from 0 to 76 hours. The software was able to count individual cells as well as confluence % at each time point by segmenting highly fluorescent objects in the images. We were able to show time-dependent ADCC killing with decrease in ZsGreen positive cells as the read-out. In addition, the uniformity of cell killing can be observed in the Celigo obtained whole well images. One of the most important findings was that there was regrowth of target cells after the initial 30 hours of cell killing, indicating the activated NK cells without antibodies did not eradicate the cancer cells. Therefore, the ability to perform long term time-course monitoring of ADCC is highly important to better characterize the effects of target antibodies on cell killing function.

HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. Large lateral surfaces involved in the DM:HLA-DR (DR) interaction have been defined, but the mechanism of catalysis is not understood. In this study, we describe four small molecules that accelerate DM-catalyzed peptide exchange. Mechanistic studies demonstrate that these small molecules substantially enhance the catalytic efficiency of DM, indicating that they make the transition state of the DM:DR/peptide complex energetically more favorable. These compounds fall into two functional classes: two compounds are active only in the presence of DM, and binding data for one show a direct interaction with DM. The remaining two compounds have partial activity in the absence of DM, suggesting that they may act at the interface between DM and DR/peptide. A hydrophobic ridge in the DMβ1 domain was implicated in the catalysis of peptide exchange because the activity of three of these enhancers was substantially reduced by point mutations in this area.

Genome-wide analyses have shown that the MHC class II region is the principal locus that confers susceptibility to a number of human autoimmune diseases. Due to the high degree of linkage disequilibrium across the MHC, it has been difficult to dissect the contribution of individual genes to disease susceptibility. As a result, intensive efforts have been made to generate mice transgenic for human class II molecules as models of autoimmune disease. However, in every case, additional manipulations—such as immunization with Ag in adjuvant, expression of immunostimulants on target tissues, or coexpression of TCR transgenes—have been required to induce disease. In this study, we show that expression of the human HLA-DQ8 (DQA1*0301/DQB1*0302) molecule alone in three lines of transgenic nonobese diabetic murine class II-deficient (mII−/−) mice results in the spontaneous development of autoimmune myocarditis. The disease shares key features of human myocarditis and was characterized by lymphocytic infiltrates in the myocardium and cardiac myocyte destruction, circulating IgG autoantibodies against cardiac myosin heavy chain, and premature death due to heart failure. We demonstrate that myocarditis could be transferred into healthy HLA-DQ8+RAG-1−/−mII−/− nonobese diabetic recipients with lymphocytes, but not sera. It has been widely thought that autoimmune myocarditis is of infectious etiology, with the immune responses arising secondary to cardiac damage from pathogens. These studies provide direct experimental evidence that spontaneous autoimmune myocarditis can occur in the absence of infection and that expression of HLA-DQ8 confers susceptibility to this organ-specific autoimmune disease.

The MHC determines susceptibility and resistance to type 1 diabetes in humans and nonobese diabetic (NOD) mice. To investigate how a disease-associated MHC molecule shapes the T cell repertoire in NOD mice, we generated a series of tetramers from I-Ag7/class II-associated invariant chain peptide precursors by peptide exchange. No CD4 T cell populations could be identified for two glutamic acid decarboxylase 65 peptides, but tetramers with a peptide mimetic recognized by the BDC-2.5 and other islet-specific T cell clones labeled a distinct population in the thymus of young NOD mice. Tetramer-positive cells were identified in the immature CD4+CD8low population that arises during positive selection, and in larger numbers in the more mature CD4+CD8− population. Tetramer labeling was specific based on the use of multiple control tetramers, including one with a single amino acid analog peptide in which a critical TCR contact residue was substituted. The T cell population was already present in the thymus of 2-wk-old NOD mice before the typical onset of insulitis and was detected in B10 mice congenic for the NOD MHC locus, but not B10 control mice. These results demonstrate that a T cell population can expand in the thymus of NOD mice to levels that are at least two to three orders of magnitude higher than estimated for a given specificity in the naive T cell pool. Based on these data, we propose a model in which I-Ag7 confers susceptibility to type 1 diabetes by biasing positive selection in the thymus and later presenting peptides from islet autoantigens to such T cells in the periphery.

Polymorphisms in MHC class II (MHCII) genes are the strongest risk factors for the development of autoimmune diseases, such as type I diabetes (T1D). T1D is associated with allelic variants of the MHCII molecule HLA-DQ. In the nonobese diabetic (NOD) mouse, the orthologous molecule, I-Ag7, also predisposes mice to spontaneous diabetes development. To investigate the functional impact of natural MHCII polymorphisms on DM binding and T1D we introduced selected mutations in the genome of the NOD mouse by CRISPR/Cas9 genomic editing. Our approach was to first identify unique polymorphisms in the putative interface of I-Ag7, and then assess the impact of these polymorphisms on DM-mediated peptide editing in vitro. Several polymorphisms increased the interaction between I-Ag7 and DM and augmented peptide binding. The substitution of lysine (K) 40 in I-Aα with glutamic acid (E) had the strongest effect on DM editing and accelerated peptide binding more than 9-fold. Lysine at position α40 is unique to the diabetes associated I-Ag7 molecule and was therefore selected for in vivo evaluation. Homozygous and heterozygous K40E mutant mice showed increased H2-DM protein expression compared to WT littermate controls. Increased H2-DM levels were accompanied by an increase in intra- and extracellular I-Ag7 expression. This suggests that the K40E mutation promotes the interaction between H2-DM and I-Ag7, and likely, represents increased intracellular dimerization and more stable I-Ag7/peptide complexes on the cell surface. Currently, we are evaluating whether increased editing of the MHCII peptide repertoire mediated by the K40E mutation influences spontaneous diabetes development.

T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4+ T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4+ T cells with a defined MHC/peptide specificity.

The crystal structures of two human TCRs specific for a HTLV-I Tax peptide bound to HLA-A2 were recently determined, for the first time allowing a functional comparison of TCRs for which the MHC/peptide/TCR structures are known. Extensive amino acid substitutions show that the native Tax residues are optimal at each peptide position. A prominent feature of the TCR contact surface is a deep pocket that accommodates a tyrosine at position 5 of the peptide. For one of these TCRs, this pocket is highly specific for aromatic residues. In the other TCR structure, this pocket is larger, allowing many different residues to be accommodated. The CTL clones also show major differences in the specificity for several other peptide residues, including side chains that are not directly contacted by the TCR. Despite the specificity of these clones, peptides that are distinct at five or six positions from Tax11–19 induce CTL activity, indicating that substantial changes of the peptide surface are tolerated. Human peptides with limited sequence homology to Tax11–19 represent partial TCR agonists for these CTL clones. The distinct functional properties of these CTL clones highlight structural features that determine TCR specificity and cross-reactivity for MHC-bound peptides.

Copolymer 1 (Cop 1, poly (Y, E, A, K)) is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS). Cop 1 binds promiscuously, with high affinity and in a peptide-specific manner to purified MS-associated HLA-DR2 (DRB1*1501) and rheumatoid arthritis-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules. In the present work at least 95% of added Cop 1 could be bound to recombinant “empty” HLA-DR1 and -DR4, and 80% could be bound to HLA-DR2 proteins. Amino acid composition, HPLC profiles, and sequencing patterns of Cop 1 eluted by acid extraction from HLA-DR molecules were similar to those of the unseparated Cop 1. Protruding N-terminal ends of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules were then treated with aminopeptidase I, followed by elution, HPLC, and pool sequencing. In contrast to untreated or unbound Cop 1, this material exhibited distinct motifs at some positions with increases in levels of E at the first and second cycles, of K at the second and third cycles, and of Y (presumably at P1 of the bound peptide) at the third to fifth cycles, regardless of the HLA-DR molecule employed. No preference was seen at the following cycles that were mainly A. These first pooled HLA-DR binding epitopes provide clues to the components of Cop 1 that are biologically active in suppressing MS and possibly rheumatoid arthritis.

The presentation of disease relevant self-peptides is clearly essential for type 1 diabetes (T1D). General inhibition of antigen presentation, however, would severely compromise a patient's immune system. The over-expression or complete loss of the MHC-like HLA-DO (DO) protein, which binds to and modulates the peptide loading ability of HLA-DM (DM; H2-M in mice), has not been shown to cause any significant deviation in immune function. Variations in the presentation of both antigenic and self-peptides have, however, been documented. Therefore, we sought to subtly alter MHC class II (MHCII) Ag processing and presentation in non-obese diabetic (NOD) mice by over-expressing the DM-modulator, DO specifically in dendritic cells (DCs). Our studies show, that NOD mice over-expressing DO (NOD.DO) in DCs never develop T1D. DO expression in DCs, prevented the infiltration of lymphocytes into pancreatic islets (insulitis), however, NOD and NOD.DO mice had similar levels of peri-islet lymphocytic infiltration. Remarkably, DO-over expression was required in only half of NOD DCs to mediate protection. NOD.DO mice were also partially protected from T1D after challenge with diabetogenic T cells from newly diabetic NOD mice. In stark contrast to T cells from NOD mice, NOD.DO T cells did not promote the rapid development of T1D when transferred into immunocompromised NOD.scid recipients supporting that NOD.DO T cells have not been primed, most likely due to limited antigen availability. Studies are underway to determine the mechanism by which DO over-expression in NOD DCs mediates protection from T1D.