Targeted Drug Delivery by MMAE Farnesyl-Bioconjugated Multivalent Chemically Self-Assembled Nanorings Induces Potent Receptor-Dependent Immunogenic Cell Death.

Antibody-drug conjugates, nanoparticles, and liposomes have been used for anticancer drug delivery. The success of targeted killing of cancer cells relies heavily on the selectivity of the drug delivery systems. In most systems, antibodies or their fragments were used as targeting ligands. In this study, we have investigated the potential for protein-based octomeric chemically self-assembled nanorings (CSANs) to be used for anticancer drug delivery. The CSANs are composed of a DHFR-DHFR fusion protein incorporating an EGFR-targeting fibronectin and the anticancer drug MMAE conjugated through a C-terminal farnesyl azide. The anti-EGFR-MMAE CSANs were shown to undergo rapid internalization and have potent cytotoxicity to cancer cells across a 9000-fold difference in EGFR expression. In addition, anti-EGFR-MMAE CSANs were shown to induce immunological cell death. Thus, multivalent and modular CSANs are a potential alternative anticancer drug delivery platform with the capability of targeting tumor cells with heterogeneous antigen expression while activating the anticancer immune response.

Multivalent Fluorinated Nanorings for On-Cell 19F NMR.

The design of imaging agents with a high fluorine content is necessary for overcoming the challenges of low sensitivity in 19F magnetic resonance imaging (MRI)-based molecular imaging. Chemically self-assembled nanorings (CSANs) provide a strategy to increase the fluorine content through multivalent display. We previously reported an 19F NMR-based imaging tracer, in which case a CSAN-compatible epidermal growth factor receptor (EGFR)-targeting protein E1-dimeric dihydrofolate (E1-DD) was bioconjugated to a highly fluorinated peptide. Despite good 19F NMR performance in aqueous solutions, a limited signal was observed in cell-based 19F NMR using this monomeric construct, motivating further design. Here, we design several new E1-DD proteins bioconjugated to peptides of different fluorine contents. Flow cytometry analysis was used to assess the effect of variable fluorinated peptide sequences on the cellular binding characteristics. Structure-optimized protein, RTC-3, displayed an optimal spectral performance with high affinity and specificity for EGFR-overexpressing cells. To further improve the fluorine content, we next engineered monomeric RTC-3 into CSAN, η-RTC-3. With an approximate eightfold increase in the fluorine content, multivalent η-RTC-3 maintained high cellular specificity and optimal 19F NMR spectral behavior. Importantly, the first cell-based 19F NMR spectra of η-RTC-3 were obtained bound to EGFR-expressing A431 cells, showing a significant amplification in the signal. This new design illustrated the potential of multivalent fluorinated CSANs for future 19F MRI molecular imaging applications.

The Many Faces of Histidine Triad Nucleotide Binding Protein 1 (HINT1).

The histidine triad nucleotide binding protein 1 (HINT1) is a nucleoside phosphoramidase that has garnered interest due to its widespread expression and participation in a broad range of biological processes. Herein, we discuss the role of HINT1 as a regulator of several CNS functions, tumor suppressor, and mast cell activator via its interactions with multiple G-protein-coupled receptors and transcription factors. Importantly, altered HINT1 expression and mutation are connected to the progression of multiple disease states, including several neuropsychiatric disorders, peripheral neuropathy, and tumorigenesis. Additionally, due to its involvement in the activation of several clinically used phosphoramidate prodrugs, tremendous efforts have been made to better understand the interactions behind nucleoside binding and phosphoramidate hydrolysis by HINT1. We detail the substrate specificity and catalytic mechanism of HINT1 hydrolysis, while highlighting the structural biology behind these efforts. The aim of this review is to summarize the multitude of biological and pharmacological functions in which HINT1 participates while addressing the areas of need for future research.

Design of Highly Fluorinated Peptides for Cell-based 19F NMR.

The design of imaging agents with high fluorine content is essential for overcoming the challenges associated with signal detection limits in 19F MRI-based molecular imaging. In addition to perfluorocarbon and fluorinated polymers, fluorinated peptides offer an additional strategy for creating sequence-defined 19F magnetic resonance imaging (MRI) imaging agents with a high fluorine signal. Our previously reported unstructured trifluoroacetyllysine-based peptides possessed good physiochemical properties and could be imaged at high magnetic field strength. However, the low detection limit motivated further improvements in the fluorine content of the peptides as well as removal of nonspecific cellular interactions. This research characterizes several new highly fluorinated synthetic peptides composed of highly fluorinated amino acids. 19F NMR analysis of peptides TB-1 and TB-9 led to highly overlapping, intense fluorine resonances and acceptable aqueous solubility. Flow cytometry analysis and fluorescence microscopy further showed nonspecific binding could be removed in the case of TB-9. As a preliminary experiment toward developing molecular imaging agents, a fluorinated EGFR-targeting peptide (KKKFFKK-ßA-YHWYGYTPENVI) and an EGFR-targeting protein complex E1-DD bioconjugated to TB-9 were prepared. Both bioconjugates maintained good 19F NMR performance in aqueous solution. While the E1-DD-based imaging agent will require further engineering, the success of cell-based 19F NMR of the EGFR-targeting peptide in A431 cells supports the potential use of fluorinated peptides for molecular imaging.

The eIF4EBP-eIF4E axis regulates CD4+ T cell differentiation through modulation of T cell activation and metabolism.

CD4+ T cells are critical for adaptive immunity, differentiating into distinct effector and regulatory subsets. Although the transcriptional programs underlying their differentiation are known, recent research has highlighted the importance of mRNA translation in determining protein abundance. We previously conducted genome-wide analysis of translation in CD4+ T cells revealing distinct translational signatures distinguishing these subsets, identifying eIF4E as a central differentially translated transcript. As eIF4E is vital for eukaryotic translation, we examined how altered eIF4E activity affected T cell function using mice lacking eIF4E-binding proteins (BP-/-). BP-/- effector T cells showed elevated Th1 responses ex vivo and upon viral challenge with enhanced Th1 differentiation observed in vitro. This was accompanied by increased TCR activation and elevated glycolytic activity. This study highlights how regulating T cell-intrinsic eIF4E activity can influence T cell activation and differentiation, suggesting the eIF4EBP-eIF4E axis as a potential therapeutic target for controlling aberrant T cell responses.

Eradication of Heterogeneous Tumors by T Cells Targeted with Combination Bispecific Chemically Self-assembled Nanorings.

Cancer stem-like cells (CSCs) are often the root cause of refractive relapse due to their inherent resistance to most therapies and ability to rapidly self-propagate. Recently, the antigen CD133 has been identified as a CSC marker on several cancer types and αCD133 therapies have shown selective targeting against CSCs with minimal off-target toxicity. Theoretically, by selectively eliminating CSCs, the sensitivity to bulk tumor-targeting therapies should be enhanced. Previously, our laboratory has developed bispecific chemically self-assembled nanorings (CSANs) that successfully induced T-cell eradication of EpCAM-positive (EpCAM+) tumors. We reasoned that targeting both CSCs [CD133-positive (CD133+)] and the bulk tumor (EpCAM+) simultaneously using our CSAN platform should produce a synergistic effect. We evaluated αCD133/αCD3 CSANs as both a single agent and in combination with αEpCAM/αCD3 CSANs to treat triple-negative breast cancer (TNBC) cells, which express a subpopulation of CD133+ cancer stem cells and EpCAM+ bulk tumor cells. Furthermore, an orthotopic breast cancer model validated the ability of αCD133 and αEpCAM targeting to combine synergistically in the elimination of TNBC MDA-MB-231 cells. Complete tumor eradication only occurred when EpCAM and CD133 were targeted simultaneously and lead to full remission in 80% of the test mice. Importantly, the depletion and enrichment of CD133 TNBCs highlighted the role of CD133+ cancer cells in regulating tumor growth and progression. Collectively, our results demonstrate that dual targeting with bispecific CSANs can be effective against heterogenous tumor cell populations and that elimination of primary and CD133+ CSCs may be necessary for eradication of at least a subset of TNBC.

Engineering Biomimetic Trogocytosis with Farnesylated Chemically Self-Assembled Nanorings.

Inspired by the natural intercellular material-transfer process of trans-endocytosis or trogocytosis, we proposed that targeted farnesylated chemically self-assembled nanorings (f-CSANs) could serve as a biomimetic trogocytosis vehicle for engineering directional cargo transfer between cells, thus allowing cell-cell interactions to be monitored and facilitating cell-cell communications. The membranes of sender cells were stably modified by hydrophobic insertion with the targeted f-CSANs, which were efficiently transferred to receiver cells expressing the appropriate receptors by endocytosis. CSAN-assisted cell-cell cargo transfer (C4T) was demonstrated to be receptor specific and dependent on direct cell-cell interactions, the rate of receptor internalization, and the level of receptor expression. In addition, C4T was shown to facilitate cell-to-cell delivery of an apoptosis inducing drug, as wells as antisense oligonucleotides. Taken together, the C4T approach is a potentially versatile biomimetic trogocytosis platform that can be deployed as a macro-chemical biological tool for monitoring cell-cell interactions and engineering cell-cell communications.

Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides.

Human histidine triad nucleotide-binding (hHINT) proteins catalyze nucleotide phosphoramidase and acyl-phosphatase reactions that are essential for the activation of antiviral proTides, such as Sofosbuvir and Remdesivir. hHINT1 and hHINT2 are highly homologous but exhibit disparate roles as regulators of opioid tolerance (hHINT1) and mitochondrial activity (hHINT2). NMR studies of hHINT1 reveal a pair of dynamic surface residues (Q62, E100), which gate a conserved water channel leading to the active site 13 Å away. hHINT2 crystal structures identify analogous residues (R99, D137) and water channel. hHINT1 Q62 variants significantly alter the steady-state kcat and Km for turnover of the fluorescent substrate (TpAd), while stopped-flow kinetics indicate that KD also changes. hHINT2, like hHINT1, exhibits a burst phase of adenylation, monitored by fluorescent tryptamine release, prior to rate-limiting hydrolysis and nucleotide release. hHINT2 exhibits a much smaller burst-phase amplitude than hHINT1, which is further diminished in hHINT2 R99Q. Kinetic simulations suggest that amplitude variations can be accounted for by a variable fluorescent yield of the E·S complex from changes in the environment of bound TpAd. Isothermal titration calorimetry measurements of inhibitor binding show that these hHINT variants also alter the thermodynamic binding profile. We propose that these altered surface residues engender long-range dynamic changes that affect the orientation of bound ligands, altering the thermodynamic and kinetic characteristics of hHINT active site function. Thus, studies of the cellular roles and proTide activation potential by hHINTs should consider the importance of long-range interactions and possible protein binding surfaces far from the active site.

Manipulating Cell Fates with Protein Conjugates.

The homeostasis of cellular activities is essential for the normal functioning of living organisms. Hence, the ability to regulate the fates of cells is of great significance for both fundamental chemical biology studies and therapeutic development. Despite the notable success of small-molecule drugs that normally act on cellular protein functions, current clinical challenges have highlighted the use of macromolecules to tune cell function for improved therapeutic outcomes. As a class of hybrid biomacromolecules gaining rapidly increasing attention, protein conjugates have exhibited great potential as versatile tools to manipulate cell function for therapeutic applications, including cancer treatment, tissue engineering, and regenerative medicine. Therefore, recent progress in the design and assembly of protein conjugates used to regulate cell function is discussed in this review. The protein conjugates covered here are classified into three different categories based on their mechanisms of action and relevant applications: (1) regulation of intercellular interactions; (2) intervention in intracellular biological pathways; (3) termination of cell proliferation. Within each genre, a variety of protein conjugate scaffolds are discussed, which contain a diverse array of grafted molecules, such as lipids, oligonucleotides, synthetic polymers, and small molecules, with an emphasis on their conjugation methodologies and potential biomedical applications. While the current generation of protein conjugates is focused largely on delivery, the next generation is expected to address issues of site-specific conjugation, in vivo stability, controllability, target selectivity, and biocompatibility.

Multivalent, Bispecific αB7-H3-αCD3 Chemically Self-Assembled Nanorings Direct Potent T Cell Responses against Medulloblastoma.

Few therapeutic options have been made available for treating central nervous system tumors, especially upon recurrence. Recurrent medulloblastoma is uniformly lethal with no approved therapies. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3. However, due to several therapy-related toxicities and reports of tumor escape, the full potential of targeting this pan-cancer antigen has yet to be realized. Here, we designed and characterized bispecific chemically self-assembling nanorings (CSANs) that target the T cell receptor, CD3ε, and tumor associated antigen, B7-H3, derived from the humanized 8H9 single chain variable fragment. We show that the αB7-H3-αCD3 CSANs increase T cell infiltration and facilitate selective cytotoxicity of B7-H3+ medulloblastoma spheroids and that activity is independent of target cell MHC class I expression. Importantly, nonspecific T cell activation against the ONS 2303 medulloblastoma cell line can be reduced by tuning the valency of the αCD3 targeted monomer in the oligomerized CSAN. Intraperitoneal injections of αB7-H3-αCD3 bispecific CSANs were found to effectively cross the blood-tumor barrier into the brain and elicit significant antitumor T cell activity intracranially as well as systemically in an orthotopic medulloblastoma model. Moreover, following treatment with αB7-H3-αCD3 CSANs, intratumoral T cells were found to primarily have a central memory phenotype that displayed significant levels of characteristic activation markers. Collectively, these results demonstrate the ability of our multivalent, bispecific CSANs to direct potent antitumor T cell responses and indicate its potential utility as an alternative or complementary therapy for immune cell targeting of B7-H3+ brain tumors.

"Switching On" Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor.

Enzymatically driven change to the spectroscopic properties of a chemical substrate or product has been a linchpin in the development of continuous enzyme kinetics assays. These assays inherently necessitate substrates or products that naturally comply with the constraints of the spectroscopic technique being used, or they require structural changes to the molecules involved to make them observable. Here we demonstrate a new analytical kinetics approach with enzyme histidine triad nucleotide binding protein 1 (HINT1) that allows us to extract both useful kcat values and a rank-ordered list of substrate specificities without the need to track substrates or products directly. Instead, this is accomplished indirectly using a "switch on" competitive inhibitor that fluoresces maximally only when bound to the HINT1 enzyme active site. Kinetic information is extracted from the duration of the diminished fluorescence when the monitorable inhibitor-bound enzyme is challenged with saturating concentrations of a nonfluorescent substrate. We refer to the loss of fluorescence, while the substrate competes for the fluorescent probe in the active site, as the substrate's residence transit time (RTT). The ability to assess kcat values and substrate specificity by monitoring the RTTs for a set of substrates with a competitive "switch on" inhibitor should be broadly applicable to other enzymatic reactions in which the "switch on" inhibitor has sufficient binding affinity over the enzymatic product.

4Ei-10 interdiction of oncogenic cap-mediated translation as therapy for non-small cell lung cancer.

In order to suppress 5' cap-mediated translation a highly available inhibitor of the interaction between the 5' mRNA cap and the eIF4E complex has been developed. 4Ei-10 is a member of the class of ProTide compounds and has elevated membrane permeability and is a strong active chemical antagonist for eIF4E. Once taken up by cells it is converted by anchimeric activation of the lipophilic 2-(methylthio) ethyl protecting group and after that Hint1 P-N bond cleavage to N7-(p-chlorophenoxyethyl) guanosine 5'-monophosphate (7-Cl-Ph-Ethyl-GMP). Using this powerful interaction, it has been demonstrated that 4Ei-10 inhibits non-small cell lung cancer (NSCLC) cell growth. In addition, treatment of NSCLC cells with 4Ei-10 results in suppression of translation and diminished expression of a cohort of cellular proteins important to maintaining the malignant phenotype and resisting apoptosis such as Bcl-2, survivin, and ornithine decarboxylase (ODC). Finally, as a result of targeting the translation of anti-apoptotic proteins, NSCLC cells are synergized to be more sensitive to the existing anti-neoplastic treatment gemcitabine currently used in NSCLC therapy.

A Phosphoramidate Strategy Enables Membrane Permeability of a Non-nucleotide Inhibitor of the Prolyl Isomerase Pin1.

The membrane permeability of nucleotide-based drugs, such as sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy, termed "ProTide", masks the anionic phosphate through an N-linked amino ester and an O-linked aromatic phospho-ester, such that release of the active drug requires consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the ProTide approach could be deployed for non-nucleotides. Here, we demonstrate that caging of a phosphate-containing inhibitor of the prolyl isomerase Pin1 increases its permeability. Moreover, this compound was processed by both esterase and phosphoramidase activity, releasing the active molecule to bind and inhibit Pin1 in cells. Thus, Hint1 appears to recognize a broader set of substrates than previously appreciated. It seems possible that other potent, but impermeable, phosphate-containing inhibitors might likewise benefit from this approach.

Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T Cell-Mediated Antitumor Responses and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression.

Overexpression of the epidermal growth factor receptor (EGFR) on various cancers makes it an important target for cancer immunotherapy. We recently demonstrated that single-chain variable fragment-based bispecific chemically self-assembled nanorings (CSANs) can successfully modify T cell surfaces and function as prosthetic antigen receptors (PARs) allowing selective targeting of tumor antigens while incorporating a dissociation mechanism of the rings. Here, we report the generation of anti-EGFR fibronectin (FN3)-based PARs with high yield, rapid protein production, predicted low immunogenicity, and increased protein stability. We demonstrated the cytotoxicity of FN3-PARs successfully while evaluating FN3 affinities, CSAN valencies, and antigen expression levels. Using an orthotopic breast cancer model, we showed that FN3-PARs can suppress tumor growth with no adverse effects and FN3-PARs reduced immunosuppressive programmed cell death ligand-1 (PD-L1) expression by downregulating EGFR signaling. These results demonstrate the potential of FN3-PARs to direct selective T cell-targeted tumor killing and to enhance antitumor T cell efficacy by modulating the tumor microenvironment.

In Vitro and In Vivo Characterization of the Anti-Zika Virus Activity of ProTides of 2'-C-ß-Methylguanosine.

The ProTide approach has emerged as a powerful tool to improve the intracellular delivery of nucleotide analogs with antiviral and anticancer activity. Here, we characterized the anti-ZIKV (ZIKV, Zika virus) activity of two ProTides of 2'-C-ß-methylguanosine. ProTide UMN-1001 is a 2'-C-ß-methylguanosine tryptamine phosphoramidate monoester, and ProTide UMN-1002 is a 2-(methylthio)-ethyl-2'-C-ß-methylguanosine tryptamine phosphoramidate diester. UMN-1002 undergoes stepwise intracellular activation to the corresponding nucleotide monophosphate followed by P-N bond cleavage by intracellular histidine triad nucleotide binding protein 1 (Hint1). UMN-1001 is activated by Hint1 but is less cell-permeable than UMN-1002. UMN-1001 and UMN-1002 were found to be more potent than 2'-C-ß-methylguanosine against ZIKV in human-derived microvascular endothelial and neuroblastoma cells and in reducing ZIKV RNA replication. Studies with a newborn mouse model of ZIKV infection demonstrated that, while treatment with 2'-C-ß-methylguanosine and UMN-1001 was lethal, treatment with UMN-1002 was nontoxic and significantly reduced ZIKV infection. Our data suggests that anchimeric activated ProTides of 2'-C-ß-methyl nucleosides should be further investigated for their potential as anti-ZIKV therapeutics.

Engineering reversible cell-cell interactions with chemical biology.

Techniques to direct cell-cell interactions have advanced our understanding of fundamental biology and opened new avenues in tissue engineering, regenerative medicine, and immunotherapy. This is often achieved by introducing new targeting ligands to the cell membrane, which can be accomplished through both genetic and nongenetic approaches. While both offer advantages, nongenetic modifications tend to be faster to produce, innocuous to the modified cell, and potentially reversible. This chapter will outline nongenetic methods that have been used to control intercellular interactions-namely hydrophobic insertion, chemical modification, liposome fusion, metabolic engineering, and enzymatic remodeling-and provide protocols that can serve as a starting point for future applications.

Profiling of Small Ribosomal Subunits Reveals Modes and Regulation of Translation Initiation.

Translation initiation is often attributed as the rate-determining step of eukaryotic protein synthesis and key to gene expression control. Despite this centrality, the series of steps involved in this process is poorly understood. Here, we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5' UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features that regulate the recruitment and processivity of scanning ribosomes and redefine optimal initiation contexts. Our approach enables deconvoluting translation initiation into separate stages and identifying regulators at each step.

Histidine triad nucleotide-binding proteins HINT1 and HINT2 share similar substrate specificities and little affinity for the signaling dinucleotide Ap4A.

Human histidine triad nucleotide-binding protein 2 (hHINT2) is an important player in human mitochondrial bioenergetics, but little is known about its catalytic capabilities or its nucleotide phosphoramidate prodrug (proTide)-activating activity akin to the cytosolic isozyme hHINT1. Here, a similar substrate specificity profile (kcat /Km ) for model phosphoramidate substrates was found for hHINT2 but with higher kcat and Km values when compared with hHINT1. A broader pH range for maximum catalytic activity was determined for hHINT2 (pK1  = 6.76 ± 0.16, pK2  = 8.41 ± 0.07). In addition, the known hHINT1-microphthalmia-inducing transcription factor-regulating molecule Ap4 A was found to have no detectable binding to HINT1 nor HINT2 by isothermal titration calorimetry. These results demonstrate that despite differences in their sequence and localization, HINT1 and HINT2 have similar nucleotide substrate specificities, which should be considered in future proTide design and in studies of their natural function.

Repression of oncogenic cap-mediated translation by 4Ei-10 diminishes proliferation, enhances chemosensitivity and alters expression of malignancy-related proteins in mesothelioma.

Activated cap-dependent translation promotes cancer by stimulating translation of mRNAs encoding malignancy-promoting proteins. The nucleoside monophosphate Protide, 4Ei-10, undergoes intracellular uptake and conversion by Hint1 to form 7-Cl-Ph-Ethyl-GMP. 7-Cl-Ph-Ethyl-GMP is an analog of cap and inhibits protein translation by binding and sequestering eIF4E thus blocking eIF4E from binding to the mRNA cap. The effects of inhibiting translation initiation by disruption of the eIF4F complex with 4Ei-10 were examined in malignant mesothelioma (MM). In a cell-free assay system, formation of the eIF4F complex was disabled in response to exposure to 4Ei-10. Treatment of MM with 4Ei-10 resulted in decreased cell proliferation, increased sensitivity to pemetrexed and altered expression of malignancy-related proteins. In light of these findings, suppression of translation initiation by small molecule inhibitors like 4Ei-10 alone or in combination with pemetrexed represents an encouraging strategy meriting further evaluation in the treatment of MM.

The LFA-1 antagonist BIRT377 reverses neuropathic pain in prenatal alcohol-exposed female rats via actions on peripheral and central neuroimmune function in discrete pain-relevant tissue regions.

Previous reports show that moderate prenatal alcohol exposure (PAE) poses a risk factor for developing neuropathic pain following adult-onset peripheral nerve injury in male rats. Recently, evidence suggests that immune-related mechanisms underlying neuropathic pain in females are different compared to males despite the fact that both sexes develop neuropathy of similar magnitude and duration following chronic constriction injury (CCI) of the sciatic nerve. Data suggest that the actions of peripheral T cells play a greater role in mediating neuropathy in females. The goal of the current study is to identify specificity of immune cell and cytokine changes between PAE and non-PAE neuropathic females by utilizing a well-characterized rodent model of sciatic nerve damage, in an effort to unmask unique signatures of immune-related factors underlying the risk of neuropathy from PAE. Cytokines typically associated with myeloid cell actions such as interleukin (IL)-1ß, tumor necrosis factor (TNF), IL-6, IL-4 and IL-10 as well as the neutrophil chemoattractant CXCL1, are examined. In addition, transcription factors and cytokines associated with various differentiated T cell subtypes are examined (anti-inflammatory FOXP3, proinflammatory IL-17A, IL-21, ROR-γt, interferon (IFN)-γ and T-bet). Lymphocyte function associated antigen 1 (LFA-1) is an adhesion molecule expressed on peripheral immune cells including T cells, and regulates T cell activation and extravasation into inflamed tissue regions. A potential therapeutic approach was explored with the goal of controlling proinflammatory responses in neuroanatomical regions critical for CCI-induced allodynia by blocking LFA-1 actions using BIRT377. The data show profound development of hindpaw allodynia in adult non-PAE control females following standard CCI, but not following minor CCI, while minor CCI generated allodynia in PAE females. The data also show substantial increases in T cell-associated proinflammatory cytokine mRNA and proteins, along with evidence of augmented myeloid/glial activation (mRNA) and induction of myeloid/glial-related proinflammatory cytokines, CCL2, IL-1ß and TNF in discrete regions along the pain pathway (damaged sciatic nerve, dorsal root ganglia; DRG, and spinal cord). Interestingly, the characteristic anti-inflammatory IL-10 protein response to nerve damage is blunted in neuropathic PAE females. Moreover, T cell profiles are predominantly proinflammatory in neuropathic Sac and PAE females, augmented levels of Th17-specific proinflammatory cytokines IL-17A and IL-21, as well as the Th1-specific factor, T-bet, are observed. Similarly, the expression of RORγt, a critical transcription factor for Th17 cells, is detected in the spinal cord of neuropathic females. Blocking peripheral LFA-1 actions with intravenous (i.v.) BIRT377 reverses allodynia in Sac and PAE rats, dampens myeloid (IL-1ß, TNF, CXCL1)- and T cell-associated proinflammatory factors (IL-17A and RORγt) and spinal glial activation. Moreover, i.v. BIRT377 treatment reverses the blunted IL-10 response to CCI observed only in neuropathic PAE rats and elevates FOXP3 in pain-reversed Sac rats. Unexpectedly, intrathecal BIRT377 treatment is unable to alter allodynia in either Sac or PAE neuropathic females. Together, these data provide evidence that: 1) fully differentiated proinflammatory Th17 cells recruited at the sciatic nerve, DRGs and lumbar spinal cord may interact with the local environment to shape the immune responses underlying neuropathy in female rats, and, 2) PAE primes peripheral and spinal immune responses in adult females. PAE is a risk factor in females for developing peripheral neuropathy after minor nerve injury.