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Long-term survival of lung transplants lags behind other solid organs due to early onset of a fibrotic form of chronic rejection known as chronic lung allograft dysfunction (CLAD). Preventing CLAD is difficult as multiple immunologic and physiologic insults contribute to its development. Targeting fibroblast activation, which is the final common pathway leading to CLAD, offers the opportunity to ameliorate fibrosis irrespective of the initiating insult. Thy-1 is a surface glycoprotein that controls fibroblast differentiation and activation.To study the role of Thy-1 in CLAD, we utilized the minor antigen mismatched C57BL/6 (B6More severe CLAD was evident in B6Our findings suggest that the loss of Thy-1 on fibroblasts is a previously unrecognized cause of CLAD and its replacement may offer therapeutic applications for amelioration of this disease post-transplantation in the setting of infectious stress responses.

Ex vivo expansion followed by reinfusion of tumor infiltrating leucocytes (TILs) has been utilized successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine interleukin-2 (IL-2) to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 post-reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic due to its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. Here we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high affinity IL-2 receptor for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident cytotoxic lymphocytes, such as CD8(+) T cells, NK cells, and γδT cells while wild-type IL-2 provides a growth advantage for CD4(+)Foxp3(+) T cells as well as myeloid cells. OMCPmutIL-2 expanded cytotoxic lymphocytes express higher levels of tumor homing receptors such as LFA-1, CD49a and CXCR3 which correlate with TIL localization to the tumor bed after intravenous injection. Consistent with this OMCPmutIL-2 expanded TILs provided superior tumor control compared to those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D expressing cytotoxic lymphocytes rather than indiscriminate expansion of all TILs.

The rapid development of molecular biotechnology presents a curricular challenge for educators trying to provide students with relevant coursework. A comprehensive biology education should also include opportunities for students to develop intellectual and technical skills through authentic research experiences. Integrating relevant and interesting research projects into their classes, however, can be a challenging task for instructors. To address these varied demands, we redesigned our existing molecular cloning course to incorporate an independent research project assessing calcium signaling. In the revised course, students use traditional and recombination-based cloning strategies to generate bacterial and mammalian expression vectors encoding CaMPARI, a novel fluorescent calcium indicator. Bacterially-expressed CaMPARI is used in protein quantification and purification assays. Students must also design their own research project evaluating the effect of chemotherapeutic agents on calcium signaling in a mammalian system. Revised and novel labs were designed to be modular, facilitating their integration into the course over 2 years. End-of-semester student evaluations were compared between years revealing a significant difference in students' perception of the course's difficulty between years. This change in attitude highlights potential pedagogical considerations that must be examined when introducing new material and activities into existing courses. Since calcium signaling is important for cellular process across diverse species, instructors may be able to develop research projects within their respective areas of interest. Integration of authentic research experiences into the curriculum is challenging; however, the framework described here provides a versatile structure that can be adapted to merge diverse instructor interests with evolving educational needs.

NKG2D is a key component of cytotoxic antitumor and antiviral responses. Multiple viruses evade NKG2D recognition by blocking NKG2D ligand expression on infected cells. In contrast, cowpox virus targets NKG2D directly by encoding a secreted antagonist, Orthopoxvirus MHC Class I-like Protein (OMCP). We have previously reported that OMCP also binds to the orphan receptor FcRL5 on innate B cells. Here, we demonstrate that mammalian-derived, glycosylated OMCP binds NKG2D but not FcRL5. Cowpox viruses either lacking OMCP, or expressing an NKG2D-binding deficient mutant, are significantly attenuated in wild type and FcRL5-deficient mice but not NKG2D-deficient mice, demonstrating that OMCP is critical in subverting NKG2D-mediated immunityin vivo. Next we determined the structure of OMCP bound to human NKG2D. Despite a structure similar to that of host NKG2D ligands, OMCP uses a drastically different orientation for NKG2D binding. The re-orientation of OMCP is associated with dramatically higher affinity for human NKG2D and the targeted interface is highly conserved in mammalian NKG2Ds, increasing the zoonotic potential of cowpox virus. We also show that cell surface presented OMCP can trigger NKG2D effector functions equivalently to host NKG2D ligands, demonstrating that NKG2D-mediated signaling requires clustering but is insensitive to binding orientation. Thus, in contrast to TCR/MHC interactions, the docking topology of NKG2D with its ligands does not appear to regulate its activation.Author SummaryVirally infected or tumor-transformed cells display NKG2D ligands (NKG2DLs) on their cell surface, which activates NKG2D-bearing lymphocytes to kill the transformed cell. Pathogens are known to counter this by blocking NKG2DL expression and/or surface display. In contrast, some tumor cells cleave endogenous NKG2DLs creating soluble NKG2D antagonists. Unlike other viral pathogens, cowpox virus uses a strategy analogous to cancer cells by targeting NKG2D directly with a soluble, high affinity NKG2D-antagonist named OMCP. We determined that OMCP’s virulencein vivois attributed to blocking NKG2D-mediated NK cell responses with no apparent effect due to binding to other receptors or cell types. We have also determined the crystal structure of cowpox OMCP bound to human NKG2D, revealing that despite conservation of the ligand scaffolding with host NKG2DLs, the viral protein is engaged with a radically altered orientation compared to all host NKG2DLs. Our structure provides key insight into how OMCP binds with an ∼5,000-fold increased affinity compared to human NKG2DLs and show that the OMCP binding site is exceptionally conserved among primates and rodents, suggesting that the ability of OMCP to recognize this conserved interface contributes to the broad zoonotic potential of cowpox virus. Finally, we show that cell membrane-anchored OMCP can trigger equivalent NKG2D-mediated killing as host NKG2DLs, demonstrating that NKG2D signaling is insensitive to ligand binding orientation.

A recurrent theme in viral immune evasion is the sabotage of MHC-I antigen presentation, which brings virus the concomitant issue of ‘missing-self’ recognition by NK cells that use inhibitory receptors to detect surface MHC-I proteins. Here, we report that rodent herpesvirus Peru (RHVP) encodes a Qa-1 like protein (pQa-1) via RNA splicing to counteract NK activation. While pQa-1 surface expression is stabilized by the same canonical peptides presented by murine Qa-1, pQa-1 is GPI-anchored and resistant to the activity of RHVP pK3, a ubiquitin ligase that targets MHC-I for degradation. pQa-1 tetramer staining indicates that it recognizes CD94/NKG2A receptors. Consistently, pQa-1 selectively inhibits NKG2A+ NK cells and expression of pQa-1 can protect tumor cells from NK control in vivo. Collectively, these findings reveal an innovative NK evasion strategy wherein RHVP encodes a modified Qa-1 mimic refractory to MHC-I sabotage and capable of specifically engaging inhibitory receptors to circumvent NK activation.

HSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH, and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the prefusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two “faces” on gD: one for receptor binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (MAbs) into five competition communities. MAbs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays, we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other MAbs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL. IMPORTANCE Virus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

A growing arm of adoptive immunotherapy for cancer involves the isolation, expansion and reinfusion of autologous tumor infiltrating leukocytes or TILs. TILs are enriched for tumor-reactive cytotoxic cells that are rendered inactive or anergic by multiple immunosuppressive mechanisms operating in solid tumors. Their separation from the tumor microenvironment followed by ex vivo activation and expansion in the presence of T cell receptor stimulation and interleukin-2 (IL-2) has been described to reverse this dysfunction and control tumor growth in some patients. Unfortunately, a large number of patients achieve no response or clinical benefit suggesting that TIL immunotherapy could benefit from refinement and improvement. Part of the limitation involves the reliance on IL-2 to support T cell expansion both in vitro and in vivo as this cytokine can result in substantial expansion and activation of regulatory T cells (Tregs) that limit the efficacy of immunotherapy. We have recently described a novel re-targeted form of IL-2, which utilizes NKG2D rather than α chain of IL-2R to form the high affinity receptor complex for β and γ chain signal transduction. This redirected cytokine fusion protein consists of a cowpox virus encoded NKG2D ligand called orthopoxvirus major histocompatibility complex class I-like protein or OMCP along with non-IL-2R-α binding mutant form of IL-2 (OMCPmutIL-2). To evaluate the effect of NKG2D-redirected cytokine delivery on TIL expansion, we isolated TILs from progressively growing B16ova melanoma and expanded them with transient anti-CD3/CD28 stimulation in the presence of continuously replenished wild-type IL-2 or OMCPmutIL-2. Compared to wild-type IL-2 the use of OMCPmutIL-2 resulted in specific and preferential expansion of NK cells, CD8+ T cells as well as γδ T cells after 2 weeks of culture. Significantly higher expansion was observed in CD8+ T (p=0.037), antigen-specific CD8+ tetramer+ (p=0.0014), NK cells (p We conclude that the use of the NKG2D retargeted common γ-chain cytokine, called OMCPmutIL-2, facilitates expansion of multiple lineages of TIL-resident cytotoxic lymphocytes and improves adoptive transfer immunotherapy over wild-type IL-2. Our data thus suggests that retargeting stimulation away from the traditional IL-2R-α chain represents a rational approach to TIL immunotherapy. Citation Format: Anirban Banerjee, Yizhan Guo, Lea Paragas, Jacqueline Slobin, Bayan Mahgoub, Dongge Li, Sarah Hein, John Westwick, Eric Lazear, Alexander S. Krupnick. Targeted expansion of NKG2D-expressing tumor infiltrating leukocytes improves adoptive transfer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 878.

Human cytomegalovirus (HCMV) is a significant cause of disease in immune-compromised adults and immune naïve newborns. No vaccine exists to prevent HCMV infection, and current antiviral therapies have toxic side effects that limit the duration and intensity of their use. There is thus an urgent need for new strategies to treat HCMV infection. Repurposing existing drugs as antivirals is an attractive approach to limit the time and cost of new antiviral drug development. Virus-induced changes in infected cells are often driven by changes in cellular kinase activity, which led us to hypothesize that defining the complement of kinases (the kinome), whose abundance or expression is altered during infection would identify existing kinase inhibitors that could be repurposed as new antivirals. To this end, we applied a kinase capture technique, multiplexed kinase inhibitor bead-mass spectrometry (MIB-MS) kinome, to quantitatively measure perturbations in >240 cellular kinases simultaneously in cells infected with a laboratory-adapted (AD169) or clinical (TB40E) HCMV strain. MIB-MS profiling identified time-dependent increases and decreases in MIB binding of multiple kinases including cell cycle kinases, receptor tyrosine kinases, and mitotic kinases. Based on the kinome data, we tested the antiviral effects of kinase inhibitors and other compounds, several of which are in clinical use or development. Using a novel flow cytometry-based assay and a fluorescent reporter virus we identified three compounds that inhibited HCMV replication with IC50 values of 3 log decrease in virus replication. These results show the utility of MIB-MS kinome profiling for identifying existing kinase inhibitors that can potentially be repurposed as novel antiviral drugs.

Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2Rα chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2Rα. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2Rα-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2., High-affinity IL-2Rα expressed by Tregs mitigates the potential of IL-2 use in cancer therapy. Here, the authors fuse IL-2 with an NKDG2 binding domain, and show that it induces IL-2 signalling selectively in NKG2D+ cells, delaying tumour growth in mice without the side effects of conventional IL-2 therapy.

As the receptor-binding protein of herpes simplex virus (HSV), gD plays an essential role in virus entry. In its native state, the last 56 amino acids of the ectodomain C terminus (C-term) occlude binding to its receptors, herpesvirus entry mediator (HVEM) and nectin-1. Although it is clear that movement of the C-term must occur to permit receptor binding, we believe that this conformational change is also a key event for triggering later steps leading to fusion. Specifically, gD mutants containing disulfide bonds that constrain the C-term are deficient in their ability to trigger fusion following receptor binding. In this report, we show that two newly made monoclonal antibodies (MAbs), MC2 and MC5, have virus-neutralizing activity but do not block binding of gD to either receptor. In contrast, all previously characterized neutralizing anti-gD MAbs block binding of gD to a receptor(s). Interestingly, instead of blocking receptor binding, MC2 significantly enhances the affinity of gD for both receptors. Several nonneutralizing MAbs (MC4, MC10, and MC14) also enhanced gD-receptor binding. While MC2 and MC5 recognized different epitopes on the core of gD, these nonneutralizing MAbs recognized the gD C-term. Both the neutralizing capacity and rate of neutralization of virus by MC2 are uniquely enhanced when MC2 is combined with MAb MC4, MC10, or MC14. We suggest that MC2 and MC5 prevent gD from performing a function that triggers later steps leading to fusion and that the epitope for MC2 is normally occluded by the C-term of the gD ectodomain.

Glycoprotein D (gD) is the receptor binding protein of herpes simplex virus (HSV) and binds to at least two distinct protein receptors, herpesvirus entry mediator (HVEM) and nectin-1. While both receptor binding regions are found within the first 234 amino acids, a crystal structure shows that the C terminus of the gD ectodomain normally occludes the receptor binding sites. Receptor binding must therefore displace the C terminus, and this conformational change is postulated to be required for inducing fusion via gB and gH/gL. When cysteine residues are introduced at positions 37 and 302 of gD, a disulfide bond is formed that stabilizes the C terminus and prevents binding to either receptor. We speculated that if disulfide bonds were engineered further upstream, receptor binding might be separated from the induction of fusion. To test this, we made five additional double cysteine mutants, each potentially introducing a disulfide bond between the ectodomain C terminus and the core of the gD ectodomain. The two mutants predicted to impose the greatest constraint were unable to bind receptors or mediate cell-cell fusion. However, the three mutants with the most flexible C terminus bound well to both HVEM and nectin-1. Two of these mutants were impaired in cell-cell fusion and null-virus complementation. Importantly, a third mutant in this group was nonfunctional in both assays. This mutant clearly separates the role of gD in triggering fusion from its role in receptor binding. Based upon the properties of the panel of mutants we conclude that fusion requires greater flexibility of the gD ectodomain C terminus than does receptor binding.

Nonliving antiviral vaccines traditionally target proteins expressed at the surface of the virion with the hope of inducing neutralizing antibodies. Orthopoxviruses (OPVs), such as the human smallpox virus and the mouse-equivalent ectromelia virus (ECTV; an agent of mousepox), encode immune response modifiers (IRMs) that can increase virulence by decreasing the host immune response. We show that one of these IRMs, the type I interferon (IFN) binding protein (bp) of ECTV, is essential for ECTV virulence and is a natural target of the antibody response. More strikingly, we demonstrate that immunization with recombinant type I IFN bp protects mice from lethal mousepox. Collectively, our experiments have important implications for our understanding of the role of IRMs in OPV virulence and of type I IFNs in OPV infections. Furthermore, our work provides proof of concept that effective antiviral vaccines can be made to prevent disease by targeting virulence factors as an alternative to the traditional approach that attempts to prevent infection by virus neutralization.

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

The NKG2D receptor is expressed on the surface of NK, T, and macrophage lineage cells and plays an important role in antiviral and antitumor immunity. To evade NKG2D recognition, herpesviruses block the expression of NKG2D ligands on the surface of infected cells using a diverse repertoire of sabotage methods. Cowpox and monkeypox viruses have taken an alternate approach by encoding a soluble NKG2D ligand, the orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity. This approach has the advantage of targeting a single conserved receptor instead of numerous host ligands that exhibit significant sequence diversity. Here, we show that OMCP binds the NKG2D homodimer as a monomer and competitively blocks host ligand engagement. We have also determined the 2.25-Å-resolution crystal structure of OMCP from the cowpox virus Brighton Red strain, revealing a truncated MHC class I-like platform domain consisting of a beta sheet flanked with two antiparallel alpha helices. OMCP is generally similar in structure to known host NKG2D ligands but has notable variations in regions typically used to engage NKG2D. Additionally, the determinants responsible for the 14-fold-higher affinity of OMCP for human than for murine NKG2D were mapped to a single loop in the NKG2D ligand-binding pocket.

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

Herpes simplex virus entry is initiated by glycoprotein D (gD) binding to a cellular receptor, such as HVEM or nectin-1. gD is activated by receptor-induced displacement of the C-terminus from the core of the glycoprotein. Binding of HVEM requires the formation of an N-terminal hairpin loop of gD; once formed this loop masks the nectin-1 binding site on the core of gD. We found that HVEM and nectin-1 exhibit non-reciprocal competition for binding to gD. The N-terminus of gD does not spontaneously form a stable hairpin in the absence of receptor and HVEM does not appear to rely on a pre-existing hairpin for binding to gD(3C–38C) mutants. However, HVEM function is affected by mutations that impair optimal hairpin formation. Furthermore, nectin-1 induces a new conformation of the N-terminus of gD. We conclude that the conformation of the N-terminus of gD is actively modified by the direct action of both receptors.