Valine 11 and phenylalanine 13 have a greater impact on the T-cell response to citrullinated peptides than the 70-74 shared epitope of the DRB1 molecule in macaques.

OBJECTIVES: Various rheumatoid arthritis (RA) HLA-DRB-1 risk haplotypes have been regrouped under the shared epitope (SE) in position 70-74. The presence of Valine in position 11 (Val11) and phenylalanine in position 13 (Phe13) are also associated with RA, but it is impossible to differentiate their role compared with the SE since they are in strong linkage disequilibrium (LD) in humans. Similar to humans, certain macaques express the SE (H6). We analysed the effect of various DRB1 haplotypes on T-cell response to citrullinated peptides (Cit-P) in macaques. METHODS: Six H6 and six non-H6 macaques were immunized with four Cit-P. T-cell response was assessed using Interferon γ enzyme-linked immunospot. RESULTS: Animals developed a specific anti-Cit-P T-cell response. Surprisingly, H6 animals had a significantly lower T-cell response than non-H6. In macaques, the 70-74 SE and the Val11 are on separate haplotypes. Presence of Val11 was strongly associated with the anti-Cit-P T-cell response, whatever the 70-74 sequence was. This response was amplified in case of presence of Phe13. CONCLUSION: The absence of LD between Val11 and SE in macaques allowed us to demonstrate that the most important HLA positions to induce a T-cell response against Cit-P were Val11 and Phe13 and not the 70-74 SE.

Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics.

Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized ß-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.

Loss of CD4 T-cell-dependent tolerance to proteins with modified amino acids.

The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.

The immunogenicity of a novel cytotoxic T lymphocyte epitope from tumor antigen PL2L60 could be enhanced by 4-chlorophenylalanine substitution at position 1.

PIWIL2, a member of PIWI/AGO family, is expressed in germline stem cells and precancerous stem cells, but not in adult somatic cells. PIWIL2 plays an important role in tumor development. It is considered as a cancer­testis antigen (CT80). It has been reported that the spliced fragment of PIWIL2, PL2L60, was widely expressed in cancer cell lines. In this study, HLA-A2-restricted epitopes from PL2L60 were predicted by online tools. To improve the activity of the native epitope, a candidate peptide P281 with potent binding affinity was chosen to investigate the modification strategy. A series of aromatic amino acids were introduced to substitute the first residue of P281. Then, we tested the binding affinity and stability of the peptide analogs and their ability to elicit specific immune responses both in vitro and in vivo. Our results indicated that the cytotoxic T lymphocytes (CTLs) induced by [4-Cl-Phe1]P281 could elicit more potent activities than that of P281 and other analogs. The CTLs induced by this analog could lyze target cells in HLA-A2-restricted and antigen-specific manners. [4-Cl-Phe1]P281 also showed the best resistance against degradation in human serum. In conclusion, the introduction of the unnatural amino acid, 4-Cl-Phe, into the first position could enhance the activity of the native epitope to induce cytotoxic T lymphocytes. It might be a good strategy to modify other promising native epitopes. The novel epitopes identified in this study could be used as novel candidates to the immunotherapy of HLA-A2 positive patients with tumors expressing PL2L60.

Lack of the T cell-specific alternative p38 activation pathway reduces autoimmunity and inflammation.

Stimulation via the T-cell receptor (TCR) activates p38α and p38ß by phosphorylation of p38 Tyr-323 (p38(Y323)). Here we characterize knockin mice in which p38α and/or ß Tyr-323 has been replaced with Phe. We find that p38α accounts for two-thirds and p38ß the remainder of TCR-induced p38 activation. T cells from double knockin mice (p38αß(Y323F)) had defects in TCR-mediated proliferation and Th1 and Th17 skewing, the former corresponding with an inability to sustain T-bet expression. Introduction of p38α(Y323F) into Gadd45α-deficient mice, in which the alternative p38 pathway is constitutively active, reversed T-cell hyperproliferation and autoimmunity. Furthermore, p38αß(Y323F) mice had delayed onset and reduced severity of the inflammatory autoimmune diseases collagen-induced arthritis and experimental autoimmune encephalomyelitis. Thus, T cell-specific alternative activation of p38 is an important pathway in T-cell proliferation, Th skewing, and inflammatory autoimmunity, and may be an attractive tissue-specific target for intervention in these processes.

A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.

Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 µM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.

A basic phenylalanine-rich oligo-peptide causes antibody cross-reactivity.

Glycolate oxidase (GO) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) are the two enzymes that serve key functions in the photorespiration and photosynthesis of plants. A 2 kDa highly basic phenylalanine-rich oligo-peptide (BOP) binds to the surface of acidic GO via ionic and hydrophobic interactions, forming the GO-BOP complex (GC). Previously, RubisCO was thought to exist as a single species composed of a large (rbc L, 54 kDa) and a small subunit (rbc S, 14 kDa). Here we show for the first time, using 2-DE, SDS-PAGE, immunoassays and amino acid determination, that BOP also interacts with RubisCO and that many RubisCO-BOP complexes (RCs), differing in pI, hydrophobicity and activity, coexist in green leaves. GCs, RCs and crude extract from green leaves analyzed by SDS-PAGE Western blotting showed that BOP exists either in subunit-BOP complexes (GO subunit-BOP, rbc L-BOP and rbc S-BOP etc.), with a wide variation in the number and the position of BOPs bound to each subunit molecular, or alone without a binding partner. When rbc L-BOP and rbc S-BOP were assayed by SDS-PAGE, BOP was dissociated from the subunit and it self-assembled to form 37 different BOP polymers (basic phenylalanine-rich protein) whose molecular weights (M(r)s) ranged from 34.0 to 91.6 kDa, indicating that the M(r) of BOP is about 2 kDa. Thus, the addition of BOP changes the M(r) of the subunit-BOP complexes so minimally that the rbc L and rbc S run at their predicted M(r)s on SDS-PAGE. In summary, the results described here demonstrate that the presence of BOP in complexes (both subunit-BOP complex and protein-BOP complex) can cause cross-reactivity of antibodies against different proteins.

Spermine and Spermidine Priming against Botrytis cinerea Modulates ROS Dynamics and Metabolism in Arabidopsis.

Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H2O2 after treatment but also after infection with B. cinerea. Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea. The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea, opening the door for the development of sustainable alternatives for chemical pesticides.

Genetic control of a shared idiotype among antibodies directed to distinct specificities.

We developed an idiotypic radioimmunoassay system that detects shared idiotypic determinants, termed GTGL idiotype, on antibodies bearing distinct antigen-binding specificities in various mouse strains. Either poly-(Glu, Tyr) (GT)- or poly-(Glu, Lys) (GL)-related determinants are able to induce anti-GT and anti-GL (GTGL)-idiotypic antibodies. Strain distribution studies indicate that GTGL-idiotypic antibodies are readily induced and frequently expressed in antisera obtained from 25 different mouse strains immunized either with GT-related or GL-related polymers. The ability to express GTGL-idiotypic antibodies is a dominant trait and is controlled by Igh-linked gene(s). In addition, we demonstrated that in anticopolymer of L-glutamine acid60- L-alanine30-L-tyrosine (GAT) and anticopolymer of L-glutamic acid54-L-lysine35-L-phenylalanine11(GLphi) antisera, both antibodies uniquely specific to GAT or GLphi, respectively, and antibodies bearing dual specificities for GAT and GLphi, expressed GTGL idiotype. The genetic implications of these findings are discussed. X

Gene complementation in the T-lymphocyte proliferative response to poly (Glu55Lys36Phe9)n. A demonstration that both immune response gene products must be expressed in the same antigen-presenting cell.

The immune response (Ir) to the random copolymer GLphi depends upon the function of two Ir genes, Ir-GLphi-beta[beta] and Ir-GLphi-alpha[alpha], mapped to the I-A and I-E/C subregions of the major histocompatibility complex, respectively. In this paper, the site(s) of expression of the products of these two Ir genes was examined by evaluating T-lymphocyte proliferative responses of bone marrow radiation chimeras. Chimeras were created in [alpha+beta- X alpha-beta+]F1 responder mice by lethal irradiation and reconstitution with a mixture of bone marrow cells from both parental strains. These chimeras failed to respond to GLphi, although they were capable or responding to the much weaker antigens, (T,G)-A--L, TEPC-15, pigeon cytochrome c, and (H,G)-A--L. This failure to respond to GLphi was shown not to be the result of a cryptic mixed lymphocyte reaction, as similar chimeras created in (alpha+beta+ X alpha-beta+)F1 mice responded well to GLphi, although they possessed almost the same potential histoincompatibility. Furthermore, the lack of response to GLphi could not be attributed to a general failure of the two parental cell types in the chimeras to collaboratc with each other, as each chimeric parental cell type could respond to dinitrophenyl conjugated ovalbumin presented on nonimmune spleen cells from the other parent. Thus, the failure of low responder parental into F1 high responder chimeras to generate an immune response to GLphi suggests that immune competence for this antigen requires at least one cell type in the immune system to express gene products of both the Ir-glphi-alpha and -beta genes, i.e. one cell must be of high responder genotype. The the antigen-presenting cell is one such cell type was shown by experiments in which GLphi-primed T lymphocytes from responder F1 mice were stimulated with antigen bound to nonimmune spleen cells. Only spleen cells from responder F1 and recombinant mice could present GLphi. Neither of the two complementing nonresponder parental spleen cell populations, either alone or mixed together, could present GLphi, although both could present purified protein derivative of tuberculin. This was shown to be the case for T cells positively selected in vitro as well as freshly explanted T cells. Thus, both Ir-GLphi-alpha and Ir-GLphi-beta gene products must be expressed in the same antigen-presenting cell to generate a T-lymphocyte proliferative response to GLphi. The implications of these findings for models of two gene complementation are discussed.

The requirement for two complementing Ir-GLphi immune response genes in the T-lymphocyte proliferative response to poly-(Glu53Lys36Phe11).

The antibody response to poly-(Glu53Lys36Phe11) (GLphi) has been shown to be under the control of two independent, major histocompatibility-linked immune response genes, designated alpha and beta. In the present work we demonstrate that the T-lymphocyte proliferative response is also under the control of these two immune response genes. Thus, mice of the H-2a, H-2b, H-2k, and H-2s haplotypes were all nonresponders to GLphi. In contrast F1 hybrids between these strains, such as (B10 X B10.A)F1 and (C3H X SJL)F1, as well as several recombinant mice derived from the nonresponder haplotypes, such as B10.1(5R), B10.HTT, and B10.S(9R), were all responders to GLphi. The complementation between nonresponder genomes appeared to be stronger in the cis position than in the trans position for some strain combinations. The failure of strains bearing only one of the two responder alleles to show a T-lymphocyte proliferative response to GLphi, argues strongly that neither gene can be expressed exclusively in B lymphocytes. This conclusion is discussed in relation to another two gene model which has recently been proposed.

Control of t-lymphocyte and B-lymphocyte activation by two complementing Ir-GLphi immune response genes.

The possibility that the two complementing alpha- and beta-Ir-GLphi genes are independently responsible for controlling events in T lymphocytes and B lymphocytes, respectively, has been tested in double adoptive transfer experiments utilizing cells from appropriate inbred strains of mice. The results of these studies show that the functions of T lymphocytes and B lymphocytes and the cooperative interactions between T and B cells require the presence of both alpha- and beta-genes in each respective cell type. Moreover, evidence has been obtained in these studies that indicates a preference for the alpha- and beta-Ir-GLphi genes in the cis position to obtain the most effective T-B-cell interactions. The possible implications of these findings are discussed.

Computational structural analysis of an anti-L-amino acid antibody and inversion of its stereoselectivity.

The binding site of a monoclonal anti-L-amino acid antibody (anti-L-AA) was modeled using the program SWISS-MODEL. Docking experiments with the enantiomers of phenylalanine revealed that the antibody interacts with L-phenylalanine via hydrogen bonds and hydrophobic contacts, whereas the D-enantiomer is rejected due to steric hindrance. Comparison of the sequences of this antibody and an anti-D-amino acid antibody (anti-D-AA) indicates that both immunoglobulins derived from the same germline progenitor. Substitution of four amino acids residues, three in the framework and one in the complementarity determining regions (CDRs), allowed in silico conversion of the anti-L-AA into an antibody that stereoselectively binds D-phenylalanine.

De novo generation of specific human IgGs by in vitro immunization using autologous proteins containing immunogenic p-nitrophenylalanine.

In vitro immunization can to used to produce monoclonal antibodies(mAbs), but this technology is limited by poor reproducibility and the requirement of pre-immunized lymphocytes. To improve this approach, we recently developed a method for rapid generation of antigen-specific B cells. Here, we report the application of this system to the production of human IgGs against tumor necrosis factor (TNF). We expressed mutant proteins with site-specific incorporated p-nitrophenylalanine (pNO2Phe), which stimulated an in vitro immune response in human immune cells. After constructing an antigen-specific antibody library from in vitro immunized B cells identified by fluorescence-activated cell sorting, we demonstrated that many point mutation events of the variable region occurred in our step-by-step co-cultivation system for affinity maturation in vitro. To mimic the class switching, we panned for high-affinity antigen-binding fragments by the phage display method, assembled them and identified hTNF-neutralizing human IgGs. This approach may provide a general method for raising high-affinity monoclonal antibodies against self-proteins. Furthermore, it supports mechanistic understanding in breaking human self-tolerance with pNO2Phe.

Efficient Acquisition of Fully Human Antibody Genes against Self-Proteins by Sorting Single B Cells Stimulated with Vaccines Based on Nitrated T Helper Cell Epitopes.

Single B cell antibody technology is a method for isolating antigen-specific B cells from human peripheral blood and obtaining antibody genes in developing antibody drugs. However, owing to immune tolerance to autoantigen, human autoantigen-specific B cells are difficult to acquire by conventional single B cell technology. In this study, we constructed a nitrated T-cell epitope named NitraTh by incorporating p-nitrophenylalanine into a universal T helper epitope. NitraTh had enhanced ability to activate CD4+ T cells and can be recognized by CD4+ T cells with different HLA class II haplotypes. This NitraTh can also break immune tolerance to autoantigens, such as human epidermal growth factor receptor 2 (HER2) and cannabinoid receptor 1, and induce strong specific IgM+ B cell responses in vitro. HER2-NitraTh vaccine can also stimulate the generation of HER2-specific IgG+ B cells in human immune system mice, which was established by cotransplanting lymphocytes and autologous dendritic cells in immunodeficient mice. We obtained 30 fully human IgG antibody genes by sorting single B cells from the human immune system mice immunized with HER2-NitraTh vaccine. The analysis of antibody genes showed that sorted B cells underwent the extensive somatic mutation of the antibody genes. We randomly selected eight genes for cloning, six of which expressed antibodies that can bind to HER2. Hence, we provided a convenient and effective method in acquiring fully human antibody genes against self-proteins, which can be used in developing therapeutic antibody drugs.

Lys89, Lys90, and Phe91 are critical core amino acid residues of the Pen ch 18 major fungal allergen recognized by human IgE antibodies.

A vacuolar serine protease (Pen ch 18) has been identified as a major allergen of Penicillium chrysogenum. The molecular features of antigenic determinant(s) on Pen ch 18 recognized by human IgE antibodies, however, have remained unclear. Here, we show that a dominant IgE epitope on the N-terminally processed Pen ch 18 allergen was narrowed down to residues 83-91. In addition, Lys89, Lys90, and possibly Phe91 were identified as the core residues. Substitution of Lys89, Lys90, or Phe91 with alanine can significantly reduce IgE-binding to Pen ch 18. Immunoblot inhibition confirmed that Lys89 and Phe91 played a significant role in IgE-binding against Pen ch 18. Molecular modeling suggests they are located on a loop-like structure at or near the surface of the major fungal allergen.

Association of immune response with efficacy and safety outcomes in adults with phenylketonuria administered pegvaliase in phase 3 clinical trials.

BACKGROUND: This study assessed the immunogenicity of pegvaliase (recombinant Anabaena variabilis phenylalanine [Phe] ammonia lyase [PAL] conjugated with polyethylene glycol [PEG]) treatment in adults with phenylketonuria (PKU) and its impact on safety and efficacy. METHODS: Immunogenicity was assessed during induction, upward titration, and maintenance dosing regimens in adults with PKU (n = 261). Total antidrug antibodies (ADA), neutralizing antibodies, immunoglobulin (Ig) M and IgG antibodies against PAL and PEG, IgG and IgM circulating immune complex (CIC) levels, complement components 3 and 4 (C3/C4), plasma Phe, and safety were assessed at baseline and throughout the study. Pegvaliase-specific IgE levels were measured in patients after hypersensitivity adverse events (HAE). FINDINGS: All patients developed ADA against PAL, peaking by 6 months and then stabilizing. Most developed transient antibody responses against PEG, peaking by 3 months, then returning to baseline by 9 months. Binding of ADA to pegvaliase led to CIC formation and complement activation, which were highest during early treatment. Blood Phe decreased over time as CIC levels and complement activation declined and pegvaliase dosage increased. HAEs were most frequent during early treatment and declined over time. No patient with acute systemic hypersensitivity events tested positive for pegvaliase-specific IgE near the time of the event. Laboratory evidence was consistent with immune complex-mediated type III hypersensitivity. No evidence of pegvaliase-associated IC-mediated end organ damage was noted. INTERPRETATION: Despite a universal ADA response post-pegvaliase administration, adult patients with PKU achieved substantial and sustained blood Phe reductions with a manageable safety profile. FUND: BioMarin Pharmaceutical Inc.

Preparation of polyclonal antibodies for nateglinide (NTG) and development of a sensitive chemiluminescent immunoassay to detect NTG in tablets and serum.

In this study, we prepared polyclonal antibodies against anti-diabetic drug nateglinide (NTG), and established a sensitive chemiluminescent immunoassay (CLIA) to detect NTG in tablets and serum. Two kinds of immunogens were synthesized using ethylcarbodiimide (EDC)/hydroxysuccinimide (NHS) and carbonyldiimidazole (CDI)/4-dimethylaminopyridine (DMAP) as coupling reagents respectively. When activated by EDC/NHS, more molecules of NTG coupled with carrier protein in immunogens. A horseradish peroxidase (HRP)-luminol-H2O2 system with p-iodophenol enhancement was applied in the CLIA analysis. The antibodies in EDC/NHS group showed higher titer, sensitivity and wider detection linear range than those in CDI/DMAP group, and were chosen for next studies. The developed CLIA assay exhibited good selectivity towards NTG among structually similar analogs. The method could detect as low as 0.35 ng mL(-1) NTG in buffer, 2.1 ng mL(-1) NTG in serum and 0.84 ng mL(-1) NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400 µg mL(-1). The CLIA method could detect 78 samples in one assay, and the samples need only dilution in pretreatment. As a summary, this research offers a sensitive assay for high-throughout screening of NTG in formulation control and pharmacokinetic studies.

Lifitegrast: First LFA-1/ICAM-1 antagonist for treatment of dry eye disease.

Dry eye disease is an extremely common condition affecting millions worldwide. The underlying pathophysiological mechanism is thought to be localized inflammation of the ocular surface resulting in the localization of T cells at this surface followed by their activation and subsequent liberation of cytokines. This effect on T cells results from the binding of lymphocyte function-associated antigen-1 (LFA-1) located on T cells to intercellular adhesion molecule 1 (ICAM-1) expressed on inflamed epithelium and endothelium, and on T cells. Lifitegrast is a T-cell integrin antagonist designed to mimic ICAM-1, thus blocking the interaction of LFA-1 and ICAM-1. Lifitegrast enters the systemic circulation to a limited extent thus reducing the likelihood of unwanted systemic reactions. Clinical trials in over 2,500 subjects with dry eye disease have shown that 5.0% lifitegrast given by ocular instillation causes a significant reduction in objective and subjective signs and symptoms of the disease. These beneficial effects are associated with a relatively low incidence of unwanted effects, almost all local in nature. In light of these findings, lifitegrast was approved by the Food and Drug Administration (FDA) in 2016 for the treatment of dry eye disease, the first drug with this mechanism of action to be so approved.