Citations

Abstract Ligation of MHC class I-recognizing receptors on NK cells dramatically modulates their secretory and cytotoxic function. This study focuses on characterizing key signaling events regulating these activities after ligation of the C-type lectin superfamily member, CD94. We isolated separate clonal populations of human NK cells in which ligation of CD94 (kp43) either triggered cell-mediated cytotoxicity (group A clones) or potently inhibited NK cell activation (group B clones). We then evaluated the proximal signaling events that regulate these alternative responses. CD94 stimulation of group A clones induced the rapid activation of intracellular protein tyrosine kinases (i.e., lck and ZAP-70), phospholipase C, and phosphatidylinositol 3-kinase. In contrast, CD94 ligation on group B clones had none of the above noted effects and instead inhibited the FcR-induced tyrosine phosphorylations of ZAP-70 and phospholipase C-gamma 2, the formation of phospho-zeta/ZAP-70 complexes, and the release of inositol phosphates. These results define distinct proximal signaling events initiated after CD94 ligation and suggest that clonotypic differences in signaling generate fundamentally different NK cell-mediated responses.

Abstract Although NK cells can kill both malignant cells and virus-infected cells without prior sensitization, it has remained unclear whether the mechanism by which an NK cell is activated in the presence of a tumor cell is similar to that induced by the presence of a virus-infected cell. In our experimental system using homogeneous populations of cloned human CD16+ NK cells, we found that HSV-infected target cells do not induce in the NK cells the same pharmacologically-active second messengers elicited by NK-sensitive tumor cells. Although phosphoinositide turnover and calcium signaling were generated in NK cells exposed to NK-sensitive tumor cells, the recognition of HSV-infected cells by NK cells did not result in similar transmembrane signaling. Furthermore, depending on the cell type infected by HSV, alternative mechanisms of cytotoxicity were employed. HSV-infected foreskin fibroblasts were rapidly and selectively killed by cloned NK cells without a requirement for IFN or accessory cells. In contrast to this direct cytotoxicity against HSV-infected foreskin fibroblasts, NK cell-mediated cytotoxicity against an HSV-infected fibrosarcoma cell line (1591) was dependent on IFN-alpha production by accessory cells. Importantly, in both systems of cytotoxicity, IFN-alpha activation of NK cells resulted in augmented killing against both infected and uninfected targets. These results suggest that NK cell activation induced during antiviral immunity is distinct from activation elicited during an antitumor response. These differences include the utilization of alternative forms of signal transduction and alternative mechanisms of cytotoxicity.

Abstract NK cells are a subpopulation of lymphocytes that kill virally infected cells and tumor cells without previous sensitization. Although exposure to distinct cytokines, including IL-2 and IL-12, can enhance these cytotoxic responses, the mechanism of this lymphokine-augmented killing remains unclear. Inasmuch as the cytotoxic event is a multistep process, there are many potential targets for lymphokine regulation. We focused on whether selected lymphokines directly modulate the intracellular signaling pathways critical for NK cell secretory function. In our experimental model, homogeneous, cloned human CD16+/CD3- NK cells were pretreated with either IL-2 or IL-12 and then stimulated with direct pharmacologic activators of the secretory response (e.g., PMA and ionomycin for intact cells or GTP gamma S for streptolysin-O permeabilized cells). Previous exposure of the cells to IL-2 or IL-12 enhanced the stimulus-induced release of granule-derived proteins (hexosaminidase and serine proteases) in a cytokine concentration- and time-dependent fashion. Furthermore, the cytokines increased the efficacies without changing the potencies of the secretagogues used in these studies. These results suggest that IL-2 and IL-12 augment NK cell-mediated cytotoxicity by increasing the maximal level of granule exocytosis evoked by Ca2+ and/or G protein-dependent intracellular signaling pathways.

Abstract Impaired cell-mediated immunity predisposes individuals to severe systemic HSV infections. A potential approach for enhancing antiviral immunity is to alter the specificity of T cells and NK cells so that they become cytotoxic against HSV. We describe here the use of heteroconjugate antibodies to augment the killing of HSV-infected cells. Two different types of heteroconjugate antibodies were used: 1) CD3-specific mAb, covalently linked to HSV-specific mAb (e.g., anti-CD3 x anti-HSV-1 glycoprotein C); 2) FcR-specific mAb linked to HSV-specific mAb (e.g., anti-Fc gamma RIII x anti-HSV-1 glycoprotein D). Whereas freshly isolated, PBL were not cytotoxic against HSV-infected target cells in a 5-h 51Cr-release assay, co-incubation with either heteroconjugate resulted in significant cytotoxicity. In vitro activated PBL (anti-CD3 + IL-2) also became more potent killers of HSV-infected cells in the presence of each heteroconjugate. The specificity of anti-CD3 x anti-HSV-1 and anti-Fc gamma RIII x anti-HSV-1 gD for enhancing T cell and NK cell immunity, respectively, was confirmed by using cloned, homogeneous human T cell and NK cell lines as effectors. Kinetic analysis demonstrated that as soon as the infected cells began to express HSV glycoproteins on their surface they became susceptible to this enhanced killing. Prolonged culture of HSV-infected cells with heteroconjugate antibodies and effector cells also decreased the amount of viral replication that occurred, as measured in a plaque inhibition assay. These results suggest that heteroconjugate antibodies are potent immunotherapeutic tools that enhance anti-HSV immunity.

Abstract NK cells mediate their cytotoxicity against tumor cells through abroad array of cytotoxic and cytostatic proteins. We investigated whether specific proteins could also be identified that contributed to NK cell-mediated antiviral immunity. Human CD16+/CD3- NK cells were obtained by using FACS and subsequently cloned by using limiting dilution. These NK cell lines, which were cytotoxic against NK-sensitive tumor targets and virally infected cells, also generated supernatants that selectively killed vesicular stomatitis virus-infected cells while sparing noninfected cells. This soluble antiviral activity was completely neutralized by antibodies specific for TNF and lymphotoxin. Purified human rTNF also duplicated this specific cytotoxicity against vesicular stomatitis virus-infected cells, as well as against CMV-, Theiler's murine encephalomyelitis virus-, and HSV-infected cells. The degree of cytotoxicity varied for the different viruses and depended on the cell type infected. These results suggest that NK cells can mediate selective and direct cytotoxicity against virally infected cells by the secretion of TNF and lymphotoxin.

AbstractPurposeEmerging literature has detailed the safe use of cefazolin in patients with immunoglobulin E–mediated penicillin allergy labeling (PAL) such as hives and anaphylaxis. The purpose of this article is to detail efforts led by an antimicrobial stewardship pharmacist working with an interdisciplinary team to optimize preoperative antimicrobials in patients with PAL.MethodsA pharmacist-led, interdisciplinary collaborative practice agreement (CPA) was activated in January 2020 to permit pharmacists to independently optimize preoperative antibiotics to the preferred cefazolin in patients with PAL if nonsevere or severe reactions had been reported. A patient registry was established covering the timeframe between January 8, 2020, and January 6, 2022. Reaction during surgery was assessed via 2-provider documentation, which included surgeon and anesthesiology staff documentation of any complications during the procedure related to a suspected allergic safety event. Utilization of cefazolin, clindamycin, and vancomycin for preoperative prophylaxis was monitored before and after implementation of the CPA.ResultsDuring the stated timeframe, 10,182 procedures and/or surgeries were completed on 1,572 (15.4%) patients with PAL and 659 (41.9%) patients previously reporting at least one reaction categorized as a severe reaction, which was hives for 71.2% of these patients. Of the 659 patients with PAL reporting a severe reaction, 356 received a preoperative cephalosporin (cefazolin, 98.8%; ceftriaxone, 1.2%) and tolerated it without a reported safety event, including 52 patients with PAL previously reporting anaphylaxis. An increase in preferred preoperative antimicrobial prophylaxis utilization was noted (cefazolin: 86% to 96.3%, P < 0.001; 2019 to 2021) with reductions noted in the use of nonpreferred preoperative antibiotics (clindamycin: 2.1% to 0.2%, P < 0.001; vancomycin: 3.2% to 0.4%, P < 0.001; 2019 to 2021).ConclusionA pharmacist-led, interdisciplinary CPA increased preferred preoperative antimicrobial use in patients with PAL reporting severe allergic reactions, including hives and anaphylaxis, without reported safety events.

ImportanceCytokine storm due to COVID-19 can cause high morbidity and mortality and may be more common in patients with cancer treated with immunotherapy (IO) due to immune system activation.ObjectiveTo determine the association of baseline immunosuppression and/or IO-based therapies with COVID-19 severity and cytokine storm in patients with cancer.Design, Setting, and ParticipantsThis registry-based retrospective cohort study included 12 046 patients reported to the COVID-19 and Cancer Consortium (CCC19) registry from March 2020 to May 2022. The CCC19 registry is a centralized international multi-institutional registry of patients with COVID-19 with a current or past diagnosis of cancer. Records analyzed included patients with active or previous cancer who had a laboratory-confirmed infection with SARS-CoV-2 by polymerase chain reaction and/or serologic findings.ExposuresImmunosuppression due to therapy; systemic anticancer therapy (IO or non-IO).Main Outcomes and MeasuresThe primary outcome was a 5-level ordinal scale of COVID-19 severity: no complications; hospitalized without requiring oxygen; hospitalized and required oxygen; intensive care unit admission and/or mechanical ventilation; death. The secondary outcome was the occurrence of cytokine storm.ResultsThe median age of the entire cohort was 65 years (interquartile range [IQR], 54-74) years and 6359 patients were female (52.8%) and 6598 (54.8%) were non-Hispanic White. A total of 599 (5.0%) patients received IO, whereas 4327 (35.9%) received non-IO systemic anticancer therapies, and 7120 (59.1%) did not receive any antineoplastic regimen within 3 months prior to COVID-19 diagnosis. Although no difference in COVID-19 severity and cytokine storm was found in the IO group compared with the untreated group in the total cohort (adjusted odds ratio [aOR], 0.80; 95% CI, 0.56-1.13, and aOR, 0.89; 95% CI, 0.41-1.93, respectively), patients with baseline immunosuppression treated with IO (vs untreated) had worse COVID-19 severity and cytokine storm (aOR, 3.33; 95% CI, 1.38-8.01, and aOR, 4.41; 95% CI, 1.71-11.38, respectively). Patients with immunosuppression receiving non-IO therapies (vs untreated) also had worse COVID-19 severity (aOR, 1.79; 95% CI, 1.36-2.35) and cytokine storm (aOR, 2.32; 95% CI, 1.42-3.79).Conclusions and RelevanceThis cohort study found that in patients with cancer and COVID-19, administration of systemic anticancer therapies, especially IO, in the context of baseline immunosuppression was associated with severe clinical outcomes and the development of cytokine storm.Trial RegistrationClinicalTrials.gov Identifier: NCT04354701