Despite advances in understanding the metabolic mechanisms of ferroptosis, the molecular events following lipid peroxide accumulation on the plasma membrane (PM) remain unclear. Herein, we identify TMEM16F as a ferroptosis suppressor at the executional phase. TMEM16F-deficient cells display heightened sensitivity to ferroptosis. Mechanistically, TMEM16F-mediated phospholipids (PLs) scrambling orchestrates extensive remodeling of PM lipids, translocating PLs at the lesion sites to reduce membrane tension, therefore mitigating the membrane damage. Unexpectedly, failure of PL scrambling in TMEM16F-deficient cells leads to lytic cell death, exhibiting PM collapse and unleashing substantial danger-associated molecule patterns. TMEM16F-deficient tumors exhibit decelerated progression. Notably, lipid scrambling inhibition synergizes with PD-1 blockade to trigger robust tumor immune rejection. The antiparasitic drug ivermectin enhances the responsiveness to PD-1 blockade by suppressing TMEM16F. Our findings uncover TMEM16F-mediated lipid scrambling as an anti-ferroptosis regulator by relocating PLs on the PM during the final stages of ferroptosis. Targeting TMEM16F-mediated lipid scrambling presents a promising therapeutic strategy for cancer treatment.

Biosynthesis drives the cell volume increase during T cell activation. However, the contribution of cell volume regulation in TCR signaling during T lymphoblast formation and its underlying mechanisms remain unclear. Here we show that cell volume regulation is required for optimal T cell activation. Inhibition of VRACs (volume-regulated anion channels) and deletion of leucine-rich repeat-containing protein 8A (LRRC8A) channel components impair T cell activation and function, particularly under weak TCR stimulation. Additionally, LRRC8A has distinct influences on mRNA transcriptional profiles, indicating the prominent effects of cell volume regulation for T cell functions. Moreover, cell volume regulation via LRRC8A controls T cell-mediated antiviral immunity and shapes the TCR repertoire in the thymus. Mechanistically, LRRC8A governs stringent cell volume increase via regulated volume decrease (RVD) during T cell blast formation to keep the TCR signaling molecules at an adequate density. Together, our results show a further layer of T cell activation regulation that LRRC8A functions as a cell volume controlling "valve" to facilitate T cell activation.

Lymphocyte decline, particularly the depletion of NK cells, is a prominent feature of immunosuppression following severe tissue injury, heightening the susceptibility of severe trauma patients to life-threatening infections. Previous research indicates that the reduction in the number of NK cells is closely associated with the process of cell death. Nonetheless, the precise mechanism of NK cell death remains unknown. Here, we discovered that following severe traumatic injury, NK cells undergo several cell death pathways, dominated by apoptosis and pyroptosis with coexistence of necrotic cell death, immunogenic cell death, ferroptosis, and autophagy. These NK cells with different paradigms of death have diverse cytokine expression profiles and diverse interactions with other immune cells. Further exploration revealed that hypoxia was strongly associated with this diverse paradigm of NK cell death. Detailed investigation of paradigms of cell death may help to enhance comprehension of lymphopenia post-severe trauma, to develop new strategy in preventing immunosuppression, and then to improve outcome for severe trauma population.

Infection by bacteria leads to tissue damage and inflammation, which need to be tightly controlled by host mechanisms to avoid deleterious consequences. It is previously reported that TMEM16F, a calcium-activated lipid scramblase expressed in various immune cell types including T cells and neutrophils, is critical for the control of infection by bacterium Listeria monocytogenes (Lm) in vivo. This function correlated with the capacity of TMEM16F to repair the plasma membrane (PM) damage induced in T cells in vitro, by the Lm toxin listeriolysin O (LLO). However, whether the protective effect of TMEM16F on Lm infection in vivo is mediated by an impact in T cells, or in other cell types, is not determined. Herein, the immune cell types and mechanisms implicated in the protective effect of TMEM16F against Lm in vivo are elucidated. Cellular protective effects of TMEM16F correlated with its capacity of lipid scrambling and augment PM fluidity. Using cell type-specific TMEM16F-deficient mice, the indication is obtained that TMEM16F expressed in liver Kupffer cells (KCs), but not in T cells or B cells, is key for protection against Listeria in vivo. In the absence of TMEM16F, Listeria induced PM rupture and fragmentation of KCs in vivo. KC death associated with greater liver damage, inflammatory changes, and dysregulated liver metabolism. Overall, the results uncovered that TMEM16F expressed in Kupffer cells is crucial to protect the host against Listeria infection. This influence is associated with the capacity of Kupffer cell-expressed TMEM16F to prevent excessive inflammation and abnormal liver metabolism.

Resident memory T (Trm) cells which are specifically located in non-lymphoid tissues showed distinct phenotypes and functions compared to circulating memory T cells and were vital for the initiation of robust immune response within tissues. However, the heterogeneity in the transcriptional features, development pathways, and cancer response of Trm cells in the small intestine was not demonstrated. Here, we integrated scRNA-seq and scTCR-seq data pan-tissue T cells to explore the heterogeneity of Trm cells and their development pathways. Trm were enriched in tissue-specific immune response and those in the DUO specially interacted with B cells via TNF and MHC-I signatures. T cell lineage analyses demonstrated that Trm might be derived from the T_CD4/CD8 subset within the same organ or migrated from spleen and mesenteric lymph nodes. We compared the immune repertoire of Trm among organs and implied that clonotypes in both DUO and ILE were less expanded and hydrophilic TRB CDR3s were enriched in the DUO. We further demonstrated that Trm in the intestine infiltrated the colorectal cancer and several effector molecules were highly expressed. Finally, the TCGA dataset of colorectal cancer implied that the infiltration of Trm from the DUO and the ILE was beneficial for overall survival and the response to immune checkpoint blockade.

Although the expression of Mex3 RNA-binding family member B (MEX3B) is upregulated in human nasal epithelial cells (HNECs) predominately in the eosinophilic chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) subtype, its functions as an RNA binding protein in airway epithelial cells remain unknown. Here, we revealed the role of MEX3B based on different subtypes of CRS and demonstrated that MEX3B decreased the TGF-β receptor III (TGFBR3) mRNA level by binding to its 3′ UTR and reducing its stability in HNECs. TGF-βR3 was found to be a TGF-β2–specific coreceptor in HNECs. Knocking down or overexpressing MEX3B promoted or inhibited TGF-β2–induced phosphorylation of SMAD2 in HNECs, respectively. TGF-βR3 and phosphorylated SMAD2 levels were downregulated in CRSwNP compared with controls and CRS without nasal polyps with a more prominent downregulation in the eosinophilic CRSwNP. TGF-β2 promoted collagen production in HNECs. Collagen abundance decreased and edema scores increased in CRSwNP compared with control, again more prominently in the eosinophilic type. Collagen expression in eosinophilic CRSwNP was negatively correlated with MEX3B but positively correlated with TGF-βR3. These results suggest that MEX3B inhibits tissue fibrosis in eosinophilic CRSwNP by downregulating epithelial cell TGFBR3 expression; consequently, MEX3B might be a valuable therapeutic target against eosinophilic CRSwNP.

'Requirements for Human Natural Killer Cells' is the latest set of guidelines on human NK cells in China, jointly drafted and agreed upon by experts from the Standards Committee of Chinese Society for Cell Biology. This standard specifies requirements for the human natural killer (NK) cells, including the technical requirements, test methods, test regulations, instructions for use, labeling requirements, packaging requirements, storage and transportation requirements, and waste disposal requirements of NK cells. This standard is applicable for the quality control of NK cells, derived from human tissues, or differentiated/transdifferentiated from stem cells. It was originally released by the Chinese Society for Cell Biology on 30 August, 2022. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human NK cells for applications.

The interaction of SIRPα with CD47 represents a major mechanism for preventing macrophage phagocytosis. However, CD47-independent mechanisms are poorly defined. Here, we report a critical role of SLAM family receptors (SFRs), ubiquitously expressed on hematopoietic cells and forming homotypic interactions, in constraining macrophage phagocytosis. We found that SFR deficiency triggered macrophage phagocytosis of hematopoietic cells, leading to severe rejection of donor hematopoietic graft in recipient mice. Specific SFR members, mainly SLAMF3 and SLAMF4, were identified as "don't eat me" receptors on macrophages. These receptors inhibited "eat me" signals, such as LRP1-mediated activation of mTOR and Syk, through SH2 domain-containing phosphatases. SFRs combined with, but were independent of, CD47 to mitigate macrophage phagocytosis, and the combined deletion of SFRs and CD47 resulted in hematopoietic cytopenia in mice. This SFR-mediated tolerance was compromised in patients with hemophagocytic lymphohistiocytosis, a syndrome characterized by inappropriate phagocytosis toward hematopoietic cells. Loss of SFRs potently elicited macrophage rejection of hematopoietic tumors. Deletion of SFRs also significantly enhanced the phagocytosis of CD19-positive hematopoietic targets by the macrophages expressing the chimeric CD19 antigen receptor. Therefore, SFR-mediated inhibition of macrophage phagocytosis is critical to hematopoietic homeostasis, and SFRs may represent previously unknown targets for tumor immunotherapy.

Follicular helper T (T FH ) cells are a specialized subset of CD4 + T cells that essentially support germinal center responses where high-affinity and long-lived humoral immunity is generated. The regulation of T FH cell survival remains unclear. Here we report that T FH cells show intensified lipid peroxidation and altered mitochondrial morphology, resembling the features of ferroptosis, a form of programmed cell death that is driven by iron-dependent accumulation of lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the major lipid peroxidation scavenger and is necessary for T FH cell survival. The deletion of GPX4 in T cells selectively abrogated T FH cells and germinal center responses in immunized mice. Selenium supplementation enhanced GPX4 expression in T cells, increased T FH cell numbers and promoted antibody responses in immunized mice and young adults after influenza vaccination. Our findings reveal the central role of the selenium–GPX4–ferroptosis axis in regulating T FH homeostasis, which can be targeted to enhance T FH cell function in infection and following vaccination.

One of the hallmarks of live cells is the asymmetric distribution of lipids across their plasma membrane. Changes in this asymmetry due to lipid "scrambling" result in phosphatidylserine exposure at the cell surface that is detected by annexin V staining. This alteration is observed during cell death processes such as apoptosis, and during physiological responses such as platelet degranulation and membrane repair. Previous studies have shown that activation of NK cells is accompanied by exposure of phosphatidylserine at the cell surface. While this response was thought to be indicative of ongoing NK cell death, it may also reflect the regulation of NK cell activation in the absence of cell death. Herein, we found that NK cell activation was accompanied by rapid phosphatidylserine exposure to an extent proportional to the degree of NK cell activation. Through enforced expression of a lipid scramblase, we provided evidence that activation-induced lipid scrambling in NK cells is reversible and does not lead to cell death. In contrast, lipid scrambling attenuates NK cell activation. This response was accompanied by reduced cell surface expression of activating receptors such as 2B4, and by loss of binding of Src family protein tyrosine kinases Fyn and Lck to the inner leaflet of the plasma membrane. Hence, lipid scrambling during NK cell activation is, at least in part, a physiological response that reduces the NK cell activation level. This effect is due to the ability of lipid scrambling to alter the distribution of membrane-associated receptors and kinases required for NK cell activation.

Metastasis is the main cause of death in individuals with cancer. Immune checkpoint blockade (ICB) can potentially reverse CD8(+) cytotoxic T lymphocytes (CTLs) dysfunction, leading to significant remission in multiple cancers. However, the mechanism underlying the development of CTL exhaustion during metastatic progression remains unclear. Here, we established an experimental pulmonary metastasis model with melanoma cells and discovered a critical role for melanoma-released exosomes in metastasis. Using genetic knockdown of nSMase2 and Rab27a, 2 key enzymes for exosome secretion, we showed that high levels of effector-like tumor-specific CD8(+) T cells with transitory exhaustion, instead of terminal exhaustion, were observed in mice without exosomes; these cells showed limited inhibitory receptors and strong proliferation and cytotoxicity. Mechanistically, the immunosuppression of exosomes depends on exogenous PD-L1, which can be largely rescued by pretreatment with antibody blockade. Notably, we also found that exosomal PD-L1 acts as a promising predictive biomarker for ICB therapies during metastasis. Together, our findings suggest that exosomal PD-L1 may be a potential immunotherapy target, suggesting a new curative therapy for tumor metastasis.

In this systematic review and meta-analysis, 25 clinical trials were systematically reviewed, and meta-analysis was performed with the results of 16 trials. It was found that the risk of surgical site infection was significantly lower in patients who received perioperative immunonutrition than those given standard nutrition. Furthermore, hospital stay was significantly shorter in patients receiving immunonutrition or early enteral nutrition after surgical resection of gastric cancer. Perioperative immunonutrition also significantly reduced white blood cell counts and the level of C-reactive protein in the patients. However, neither CD4+ T cells nor inflammatory cytokines were significantly affected even though immunonutrition was in favor. These findings suggested that patients with gastrointestinal cancer may benefit from perioperative immunonutrition support by reducing surgery-associated complications and shortening hospital stay. The effects and the underlying mechanism of immunonutrition on immunological modulation and inflammatory regulation, however, remain to be further defined.

Plasma membrane damage and cell death during processes such as necroptosis and apoptosis result from cues originating intracellularly. However, death caused by pore-forming agents, like bacterial toxins or complement, is due to direct external injury to the plasma membrane. To prevent death, the plasma membrane has an intrinsic repair ability. Here, we found that repair triggered by pore-forming agents involved TMEM16F, a calcium-activated lipid scramblase also mutated in Scott's syndrome. Upon pore formation and the subsequent influx of intracellular calcium, TMEM16F induced rapid "lipid scrambling" in the plasma membrane. This response was accompanied by membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice exhibited compromised control of infection by Listeria monocytogenes associated with a greater sensitivity of neutrophils to the pore-forming Listeria toxin listeriolysin O (LLO). Thus, the lipid scramblase TMEM16F is critical for plasma membrane repair after injury by pore-forming agents.

Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, China Department of Rheumatology and Immunology, The Third Affiliated Hospital, Southern Medical University, Guangzhou, China Department of Rheumatology and Immunology, Shunde Hospital, Southern Medical University, Foshan, China Department of Pathology, Immunology and Laboratory Medicine, Rutgers University-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA Center for Immunity and Inflammation, New Jersey Medical School, Rutgers-The State University of New Jersey, Newark, NJ 07101, USA Department of Immunology and Infectious Diseases, The John Curtin School of Medical Research, The Australian National University, Acton, ACT, Australia

The ζ-associated protein of 70 kDa (ZAP-70) is a cytoplasmic tyrosine kinase critical for intracellular signaling downstream of the T-cell receptor (TcR). Biallelic mutations in ZAP70 cause combined immunodeficiency (CID). Classically, ZAP-70 deficiency results from null alleles and presents with failure to thrive and severe/recurrent infections in infancy (ie, severe CID) that is marked by absent circulating CD8+ T cells, quantitatively normal yet dysfunctional CD4+ T cells accounting for hypo-/dysgammaglobulinemia, with quantitatively intact B cells and natural killer cells and requires hematopoietic stem cell transplant for survival beyond early childhood1Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Google Scholar, 2Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 3Gelfand E.W. Weinberg K. Mazer B.D. Kadlecek T.A. Weiss A. Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.J Exp Med. 1995; 182: 1057-1065Crossref PubMed Scopus (84) Google Scholar, 4Elder M.E. Skoda-Smith S. Kadlecek T.A. Wang F. Wu J. Weiss A. Distinct T cell developmental consequences in humans and mice expressing identical mutations in the DLAARN motif of ZAP-70.J Immunol. 2001; 166: 656-661Crossref PubMed Google Scholar, 5Honig M. Schuetz C. Schwarz K. Rojewski M. Jacobsen E. Lahr G. et al.Immunological reconstitution in a patient with ZAP-70 deficiency following transfusion of blood lymphocytes from a previously transplanted sibling without conditioning.Bone Marrow Transplant. 2012; 47: 305-307Crossref PubMed Scopus (8) Google Scholar (see Table E1 in this article's Online Repository at www.jacionline.org). "Leaky" ZAP-70 deficiency has been reported only once: a 9-year-old boy with nonsevere skin and lung infections had a homozygous intronic mutation producing a novel splice acceptor site, resulting in predominant expression of a frame-shifted mutant protein but with residual levels of wild-type (WT) protein.6Picard C. Dogniaux S. Chemin K. Maciorowski Z. Lim A. Mazerolles F. et al.Hypomorphic mutation of ZAP70 in human results in a late onset immunodeficiency and no autoimmunity.Eur J Immunol. 2009; 39: 1966-1976Crossref PubMed Scopus (63) Google Scholar Recently, a sibling pair manifesting only early-onset autoimmune diseases, without opportunistic infections, was shown to be compound heterozygous for a hypoactive allele and a weakly hyperactive allele; the specific combination of these paired alleles was required for disease.7Chan A.Y. Punwani D. Kadlecek T.A. Cowan M.J. Olson J.L. Mathes E.F. et al.A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70.J Exp Med. 2016; 213: 155-165Crossref PubMed Scopus (20) Google Scholar Collectively, these cases show that residual levels of functional protein regulate the clinical phenotype of ZAP-70 deficiency. It further suggests that, in distinction to null alleles, different therapeutic approaches may be needed for "leaky" mutations that permit survival beyond the typical time frame and for whom hematopoietic stem cell transplant at older age may be associated with excess risk.8Pai S.Y. Logan B.R. Griffith L.M. Buckley R.H. Parrott R.E. Dvorak C.C. et al.Transplantation outcomes for severe combined immunodeficiency, 2000-2009.N Engl J Med. 2014; 371: 434-446Crossref PubMed Scopus (569) Google Scholar We expand the clinical phenotype of hypomorphic ZAP-70 deficiency and provide proof-of-concept that mutation-targeted therapy can improve immune function. We identified a 33-year-old male (P1) born from consanguineous Coptic parents (see Fig E1, A, in this article's Online Repository at www.jacionline.org). An older brother died at 16 months from suspected viral encephalitis; an older sister and parents were well. P1 had a history of failure to thrive, adrenal insufficiency, and inflammatory enterocolitis starting in infancy, recurrent respiratory tract infections starting at 9 months, and intermittent mucocutaneous candidiasis, recurrent herpes simplex virus stomatitis, and varicella-zoster virus infections 3 times. Investigations at the time revealed a visible thymus, normal immunoglobulin isotypes with poor response to vaccines, and persistent CD8+ lymphocytopenia (1% to 3% of total T cells age-adjusted reference range). Beginning at approximately 3 years of age, serial testing demonstrated increasing proportion of CD8+ T cells (8% to 10% of total lymphocytes). Further investigations were suggestive of ZAP-70 deficiency (Fig E1, B and C). However, a reference laboratory reported identifying no mutation in ZAP70. Thus, bone marrow transplantation was not pursued. IgA nephropathy at age 16 years eventually required renal transplant from his mother at age 30 years. The subsequent years were marked by EBV viremia/lymphoproliferative disorder (with recurrence of viremia ∼6 months after completing rituximab), cytomegalovirus viremia (with recurrence following cessation of (val)ganciclovir), polyomaviremia (BK and JC viruses), and epidermodysplasia verruciformis-like lesions (due to human papilloma virus-23) (see Fig E2 in this article's Online Repository at www.jacionline.org), which prompted reevaluation for an underlying immunodeficiency. He is currently aged 35 years, on immunoglobulin replacement for respiratory tract infection prophylaxis, and his viral infections are managed conservatively. Whole-exome sequencing revealed no known etiology for the immunodeficiency, other than a homozygous c.1272C>T variant in ZAP70 (NM_001079; hg19). The germline c.1272C>T variant, while synonymous (p.G355G), is predicted in silico (Human Splicing Finder) to create a novel donor splice site within exon 9 (DSSmut) that is stronger than the native junctional one (DSSwt) (Fig 1, A). This new splice site is predicted to create a 19-bp deleted product with a premature stop codon in exon 10. Sanger sequencing confirmed the homozygous c.1272C>T variant in P1; the unaffected family members were heterozygous (Fig 1, B). RT-PCR from P1's PBMCs confirmed the predicted splicing effect, demonstrating a predominant mutant isoform with trace levels of WT isoform (Fig 1, C and D). The heterozygous family members possessed both isoforms but with higher WT levels; unrelated healthy controls expressed only the WT isoform. Sequencing of gel-eluted bands confirmed the 19-bp deletion in the mutant isoform (data not shown). Thus, c.1272C>T variant creates a mutant mRNA isoform through aberrant splicing; in homozygous state, the mutant isoform predominates over WT in P1. To determine the impact of the predominant aberrant ZAP70 mRNA isoform on protein production, we performed immunoblot analysis on P1's PBMCs and confirmed reduced ZAP-70 levels, as was seen in the original childhood investigations (Fig E1, C). Plasmids encoding the open-reading frame of WT ZAP-70 (ZAP-70wt) or the 19-bp deleted variant (ZAP-70mut) were stably transfected into ZAP-70–deficient Jurkat P116 cells, and immunoblot of cell lysates confirmed the absence of ZAP-70 protein with the mutant isoform (Fig 1, E). TcR signaling downstream of ZAP-70, assessed by phosphorylation of linker for activation of T cells, was absent following OKT3 stimulation of ZAP-70mut–transfected Jurkat P116, but intact with ZAP-70wt (see Fig E3, B, in this article's Online Repository at www.jacionline.org). These findings confirm that the mutant ZAP70 mRNA resulting from the c.1272C>T variant is null. Although the mutant donor splice site (DSSmut) is stronger than the native WT site (DSSwt), the latter is still intact and functionally able to generate WT ZAP70 mRNA and protein. In P1's homozygous mutant state, this is clearly inadequate for T-cell homeostasis. However, in heterozygous family members, ZAP-70 WT protein is sufficiently produced to maintain an otherwise well state. We hypothesized that blocking the stronger DSSmut in P1 would favor usage of DSSwt to increase WT ZAP-70 levels and consequently, T-cell function. To prove this, we designed an antisense morpholino oligonucleotide (AMO) to target the c.1272C>T variant while sparing the native exon-intron junction so as to block the mutant splice effect during processing of ZAP70 pre-mRNA (Fig 1, F). To confirm that the AMO specifically inhibited expression of the mutant ZAP70 isoform (ie, blocked its transition from pre-mRNA to mRNA), we treated P1's PBMCs and demonstrated correction of splicing (Fig 1, G). Optimization studies demonstrated maximal corrective effect starting at 48 hours using 5 μM AMO, producing approximately 50% WT mRNA (confirmed by sequencing; data not shown). A similar AMO-induced effect on mutant mRNA was seen on the sister's cells (data not shown); there were insufficient number of cells from P1's parents for testing. To verify the specificity of this effect, we treated cells from healthy controls and observed no effect (data not shown). Immunoblot analysis of P1's AMO-treated PBMCs demonstrated increased ZAP-70 expression, confirming that the correction of aberrant splicing results in augmented production of ZAP-70 protein (Fig 2, A and B). AMO-treated PBMCs demonstrated increased linker for activation of T cells activation following OKT3 stimulation compared with untreated cells (Fig 2, A and B), indicating that this restored ZAP-70 retains functionally intact TcR signaling. To determine whether the AMO could improve cellular function, we evaluated the expression of prototypical T-cell cytokine genes, IL2, IL4, IL10, and IFNG (IFN-γ) (Fig 2, C). AMO treatment alone increased IL2, IL4, and IL10 expression; IL2 and IL4 expression was further enhanced by CD3/CD28 stimulation (Fig 2, C). Treatment with AMO had no effect on IFN-γ expression (data not shown), likely due to the reestablished TH2-driven responses in the PBMCs polarizing away from TH1 responses. Last, AMO treatment reestablished the proliferative response to CD3/CD28 costimulation in P1's primary T cells, an effect seen in both CD4+ and CD8+ T cells (Fig 2, D and E; see Fig E4 in this article's Online Repository at www.jacionline.org). Collectively, these findings demonstrate that AMO treatment increases the expression of WT ZAP-70 protein, which is molecularly intact and capable of restoring key T-cell functional responses. Analysis of this hypomorphic ZAP-70–deficient patient uniquely illustrates several key points: (1) Clinically, the morbid natural history suggests a role for ZAP-70 in the functional control of double-stranded DNA viruses beyond the generation of CD8+ T cells, which requires further elucidation. Interestingly, the autoimmune diseases here (colitis; nephropathy) were also reported by Chan et al,1Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Google Scholar who elegantly demonstrated that a fine molecular balance in ZAP-70 effect on T-cell signaling can be associated with autoimmunity only. (2) Genetically, it shows that synonymous genetic variants may be deleterious by modifying splicing, with hypomorphism resulting from a skewed net balance of amorphic versus residual WT protein. (3) Immunotherapeutically, it demonstrates that the accurate identification of causal genetic lesions permits a mutation-silencing approach that leads to molecular correction and recuperation of fundamental T- cell cytokine responses, at least in vitro. Although the breadth of T-cell functions could not be fully investigated pragmatically, to our knowledge this represents the first example in which AMO therapy successfully restored immune function in primary cells from a human with a genetically defined immunodeficiency. This approach could also extend to therapeutically modulate functional single-nucleotide polymorphisms in immune genes. Clearly, additional parameters will need to be addressed before this approach can be used in clinical practice (eg, pharmacokinetics and safety), although this strategy is in use for inborn errors of muscle or metabolism.9Winkler J. Oligonucleotide conjugates for therapeutic applications.Ther Deliv. 2013; 4: 791-809Crossref PubMed Scopus (119) Google Scholar Overall, the insight gained from this work opens new avenues for individualized, mutation-targeting therapy for CID. Subjects and the patient's family members provided informed consent on McGill University Health Centre institutional review board–approved research protocol (GEN10-256). Comprehensive medical histories, including review of all available outside records and serial clinical evaluations, and clinical immunologic laboratory testing were performed at the McGill University Health Centre. TH17 enumeration from whole blood was performed as previously described.E1Renner E.D. Rylaarsdam S. Anover-Sombke S. Rack A.L. Reichenbach J. Carey J.C. et al.Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome.J Allergy Clin Immunol. 2008; 122: 181-187Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar For whole-exome sequencing, exome enrichment was conducted on genomic DNA using the SeqCap EZ Exome v3+ UTR kit (64Mb sequence capture) from Roche-Nimblegen (Madison, Wis). The enriched genomic DNA fragments were sequenced (PE100) on an Illumina HiSeq 2000.The whole-exome sequencing data were analyzed following the GATK Best Practices recommendations for variant discovery in DNA-Seq, with GATK version 3.E2Van der Auwera G.A. Carneiro M.O. Hartl C. Poplin R. Del Angel G. Levy-Moonshine A. et al.From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline.Curr Protoc Bioinformatics. 2013; 43 (11.10. 1-33)Crossref PubMed Scopus (4200) Google Scholar, E3DePristo M.A. Banks E. Poplin R. Garimella K.V. Maguire J.R. Hartl C. et al.A framework for variation discovery and genotyping using next-generation DNA sequencing data.Nat Genet. 2011; 43: 491-498Crossref PubMed Scopus (7900) Google Scholar, E4McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. et al.The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (17508) Google Scholar, E5Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (31723) Google Scholar, E6Cingolani P. Patel V.M. Coon M. Nguyen T. Land S.J. Ruden D.M. et al.Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift.Front Genet. 2012; 3: 35Crossref PubMed Scopus (627) Google Scholar, E7A set of command line tools (in Java) for manipulating high-throughput sequencing (HTS) data and formats such as SAM/BAM/CRAM and VCF. n.d. Available at: http://broadinstitute.github.io/picard/. Accessed May 16, 2016.Google Scholar, E8Andrews S. FastQC A quality control tool for high throughput sequence data. n.d. Available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Accessed May 16, 2016.Google Scholar After verifying the quality of the reads with FastQC,E8Andrews S. FastQC A quality control tool for high throughput sequence data. n.d. Available at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Accessed May 16, 2016.Google Scholar the reads were aligned with the Burrows-Wheeler Aligner (v0.7.12-r1039)E5Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (31723) Google Scholar to the GRCh37 reference genome. Duplicate reads were marked with Picard tools.E7A set of command line tools (in Java) for manipulating high-throughput sequencing (HTS) data and formats such as SAM/BAM/CRAM and VCF. n.d. Available at: http://broadinstitute.github.io/picard/. Accessed May 16, 2016.Google Scholar Reads were realigned around indels with GATK IndelRealigner. Base quality scores were recalibrated with GATK BaseRecalibrator. Variant calling was performed with GATK HaplotypeCaller. Variant annotation was performed with SnpSiftE6Cingolani P. Patel V.M. Coon M. Nguyen T. Land S.J. Ruden D.M. et al.Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift.Front Genet. 2012; 3: 35Crossref PubMed Scopus (627) Google Scholar and SnpEff.E9Cingolani P. Platts A. Wang le L. Coon M. Nguyen T. Wang L. et al.A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.Fly (Austin). 2012; 6: 80-92Crossref PubMed Scopus (6989) Google Scholar Only variants with coverage of at least 10 reads were kept for downstream analysis. To identify rare variants, the frequency of the variants in the Exome Aggregation ConsortiumE10Lek M. Karczewski K. Minikel E. Samocha K. Banks E. Fennell T. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (7176) Google Scholar database was verified. For Sanger sequencing, the ZAP70 gene (NG_007727.1) was PCR amplified from genomic DNA using primers designed to flank the respective regions (primers and sequencing conditions available on request). Sequencing was performed at the McGill University and Génome Québec Innovation Centre. Sequencing analyses were performed on Sequencher sequence analysis software (Gene Codes Corporation, Ann Arbor, Mich). Potential splice sites were predicted using the online tool Human Splicing Finder (www.umd.be/HSF/). The Myc-Flag–tagged ZAP-70 plasmid was from OriGene (Rockville, Md). Site-directed mutagenesis was used to create a 19-bp deletion at the end of exon 9 (New England Biolabs, Whitby, Ontario, Canada). Jurkat ZAP-70–deficient (p116) Jurkat cells were stably transfected with either WT or mutant ZAP-70 plasmid by electroporation followed by G418 selection (1 mg/mL). A 20-mer AMO was designed to target the de novo mutant donor splice site generated by the c.1272C>T mutation in the pre-mRNA of ZAP70, while sparing the native donor splice site. The AMO sequence was as follows: 5′-GCATGCGGTACACACCCTGG-3′. Vivo-morpholinos were used at a concentration of 5 μM. AMOs were designed and synthesized by Gene-Tools, LLC (Philomath, Ore). PBMCs were isolated as previously described.E11Gavino C. Cotter A. Lichtenstein D. Lejtenyi D. Fortin C. Legault C. et al.CARD9 deficiency and spontaneous central nervous system candidiasis: complete clinical remission with GM-CSF therapy.Clin Infect Dis. 2014; 59: 81-84Crossref PubMed Scopus (139) Google Scholar, E12Vinh D.C. Schwartz B. Hsu A.P. Miranda D.J. Valdez P.A. Fink D. et al.Interleukin-12 receptor beta1 deficiency predisposing to disseminated Coccidioidomycosis.Clin Infect Dis. 2011; 52: e99-e102Crossref PubMed Scopus (78) Google Scholar PBMCs were cultured in Opti-MEM Reduced Serum Media (Thermo Scientific, Waltham, Mass) and stimulated with 1 μg/mL OKT3 followed by 3 μg/mL secondary antibody or added to 96-well round-bottom plates coated with 3 μg/mL anti-CD28 and 1 μg/mL OKT3. There were insufficient number of cells from the sister and parents for functional testing. Samples were acquired on a BD FACSCANTO II flow cytometer. Single-color compensation samples were prepared using AbC Total Antibody Compensation Bead Kit (Thermo Scientific). Cells were labeled with CellTrace CFSE Cell Proliferation Kit (Thermo Scientific) and LIVE/DEAD Fixable Cell Stain Kit (Thermo Scientific) as per manufacturers' instructions. Compensation and analysis were performed on FlowJo version V10.2 Samples were separated on Bis-Tris Polyacyrlamide Gels (Thermo Scientific), transferred onto polyvinylidene difluoride membranes, and immunoblotted. Band densities were measured with ImageJ software (http://imagej.nih.gov/ij/, 1997-2014). Antibodies against phosphorylated linker for activation of T cells (Tyr171), and β-actin were from Cell Signaling Technology (Danvers, Mass). Total linker for activation of T cells antibody was from Santa Cruz Biotechnology (Dallas, Texas). Anti-Flag was from OriGene. Antibodies anti-CD3 (OKT3) and anti-CD28 were from Biolegend (San Diego, Calif). Antibodies against ZAP-70, Lck, Fyn, and Csk were as described previously.E13Latour S. Chow L.M. Veillette A. Differential intrinsic enzymatic activity of Syk and Zap-70 protein-tyrosine kinases.J Biol Chem. 1996; 271: 22782-22790Crossref PubMed Scopus (137) Google Scholar, E14Davidson D. Chow L.M. Fournel M. Veillette A. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn.J Exp Med. 1992; 175: 1483-1492Crossref PubMed Scopus (133) Google Scholar Allophycocyanin-conjugated anti-human CD3 was from Thermo Scientific. Phycoerythrin-conjugated anti-human CD4 and APC-Cy7–conjugated CD8 were from BD Biosciences (Mississauga, Ontario, Canada). Total RNA was isolated using the RNeasy kit (Qiagen, Mississauga, Ontario, Canada) and reverse transcribed with the Maxima cDNA synthesis kit for RT-quantitative PCR (Thermo Scientific). Quantitative real-time PCR was performed using the Taqman quantitative PCR Gene Expression assay system with probes directed against IL2 (assay ID Hs00174114_m1), IL4 (assay ID Hs00174122_m1), IL10 (assay ID Hs00961622_m1), UBASH3A (assay ID Hs00955170_m1), and CD28 (assay ID Hs01007422_m1) (Thermo Scientific) on Applied Biosystem 7500 real-time PCR system. The mRNA input was normalized to the expression of the T-cell–specific housekeeping gene UBASH3A to account for any variability in T-lymphocyte composition. One experiment representative of 3 independent experiments performed is shown. Graphs and statistical analyses were generated with GraphPad Prism Version 6.00. Statistical significance was calculated according to an unpaired 2-tailed ratio student t test, with P < .05 considered significant.Fig E2A, Well-circumscribed pink macules on the trunk torso. B, Hematoxylin and eosin stain demonstrating enlarged superficial keratinocytes with distinctive blue-gray cytoplasm in association with prominent keratohyaline granules, consistent with HPV infection. C, Immunohistochemical pan-HPV stain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3A, A schematic representation of the ZAP-70 protein, with the asterisk denoting the location of the c.1272C>T mutation. B, Jurkat P116 cells were stimulated with OKT3 antibody and cell lysates were analyzed by immunoblot using antibodies specific for LAT and phospho-LAT. LAT, Linker for activation of T cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Gating strategy for assessment of T-cell proliferation of CFSE-labeled, CD3/CD28-stimulated cells. APC, Allophycocyanin; CFSE, carboxyfluorescein succinimidyl ester; FITC, fluorescein isothiocyanate; PE, phycoerythrin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Reports of ZAP-70 deficiencyCaseReferenceMutationEffect on proteinAge of onset; sexInfectionsAutoimmune/dysregulated inflammationImmunologic phenotypeOutcome1E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google ScholarCompound heterozygous<1 y-old M; MennoniteUpper respiratory tract Infections; Pneumonia; Oral ulcerations; PCP*Diarrhea, weight lossTotal CD3: normal.CD4+ T: normal.CD8+ T: absent.B: elevated.Immunoglobulin: IgG (↑); IgA (↑); IgM (N); IgE (NR)HLA-matched unrelated BMT at 20 mo with successful engraftment but died at 22 mo (unclear etiology)c.1763C>Ap.S518R.No protein detected in patients' cells.c.1624-11G>A [NM_001079.3]; G to A transition in intron 12 [g.98354447G>A; assembly GRCh37]Creation of stronger splicing acceptor site leading to addition of 3 amino acids (LEQ) in the catalytic domain. No protein detected in patients' cells2E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google ScholarNeonate M (brother of above case); identified by immunophenotypic screen; MennoniteNone (kept in protective isolation).(Had CMV in urine and rotavirus in stool, but was asymptomatic)NRTotal CD3: decreased.CD4+ T: normal.CD8+ T: absent.B: elevated.Immunoglobulin: NRHLA-matched unrelated BMT at 4 mo; alive at age 2 y3E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google Scholar6 mo F; Mennonite; unrelated to above casesOtitis media with perforation; PCPNRTotal CD3: normal.CD4+ T: elevated.CD8+ T: absent.B: normal.Immunoglobulin: IgG (↓); IgA (↑); IgM (N); IgE (NR)HLA-matched related BMT at 11 mo; alive at 4 y4E15Monafo W.J. Polmar S.H. Neudorf S. Mather A. Filipovich A.H. A hereditary immunodeficiency characterized by CD8+ T lymphocyte deficiency and impaired lymphocyte activation.Clin Exp Immunol. 1992; 90: 390-393Crossref PubMed Scopus (30) Google Scholar, E16Chan A.C. Kadlecek T.A. Elder M.E. Filipovich A.H. Kuo W.L. Iwashima M. et al.ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.Science. 1994; 264: 1599-1601Crossref PubMed Scopus (455) Google Scholar, E17Arpaia E. Shahar M. Dadi H. Cohen A. Roifman C.M. Defective T cell receptor signaling and CD8+ thymic selection in humans lacking zap-70 kinase.Cell. 1994; 76: 947-958Abstract Full Text PDF PubMed Scopus (507) Google Scholar, E18Roifman C.M. Hummel D. Martinez-Valdez H. Thorner P. Doherty P.J. Pan S. et al.Depletion of CD8+ cells in human thymic medulla results in selective immune deficiency.J Exp Med. 1989; 170: 2177-2182Crossref PubMed Scopus (65) Google Scholar5 mo F (sister of case 3); Mennonite; identified by immunophenotypic screenOral ulcerations with eczematous rash; PCP; chronic diarrhea with reovirus; oral thrushNRTotal CD3: normal.CD4+ T: elevated.CD8+ T: absent.B: normal.Immunoglobulin: IgG (N); IgA (N); IgM (N); IgE (NR)Partial HLA-matched unrelated BMT at age 24 mo; alive at 3 y5E19Elder M.E. Lin D. Clever J. Chan A.C. Hope T.J. Weiss A. et al.Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase.Science. 1994; 264: 1596-1599Crossref PubMed Scopus (455) Google ScholarHomozygous: 1719_1931del13 with predicted frameshiftNo protein detected in patient's cellsNRNRNRCD4+ T: normal.CD8+ T: absent.B: normal.Immunoglobulin: NRNR6E20Gelfand E.W. Weinberg K. Mazer B.D. Kadlecek T.A. Weiss A. Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.J Exp Med. 1995; 182: 1057-1065Crossref PubMed Scopus (93) Google Scholar, E21Mazer B. Harbeck R.J. Franklin R. Schwinzer R. Kubo R. Hayward A. et al.Phenotypic features of selective T cell deficiency characterized by absence of CD8+ T lymphocytes and undetectable mRNA for ZAP-70 kinase.Clin Immunol Immunopathol. 1997; 84: 129-138Crossref PubMed Scopus (13) Google ScholarMutation not defined but no mRNA detectedNo protein4.5 mo M; not MennoniteMultiple respiratory infections; laryngomalacia; enteritis due to coronavirus; pneumonitis due to human parainfluenza virus 3Diarrhea, fail

Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Phagocytosis by macrophages plays a critical role in cancer control. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo, suggesting that blockade of the SIRPα-CD47 checkpoint could be useful in treating human cancer. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα-CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα-CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα-CD47 blockade therapy.

Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

Signaling lymphocytic activation molecule (SLAM) family receptors (SFRs) can mediate either activating or inhibitory effects during natural killer cell (NK cell) activation. In this study, we addressed the global role, regulation, and mechanism of action of the SLAM family in NK cells by analyzing a mouse lacking the entire ∼400-kilobase Slam locus, which encodes all six SFRs and CD48, the ligand of SFR 2B4. This mouse displayed enhanced NK cell activation responses toward hematopoietic target cells. Analyses of mice lacking individual SFRs showed that the inhibitory function of the Slam locus was due solely to 2B4 and was not influenced positively or negatively by other SFRs. Differences in NK cell responses between recognition of targets expressing or lacking ligands for SFRs were enhanced by IL-12 but suppressed by type I interferon. Cytokines also changed the levels of SLAM-associated protein adaptors, which prevent the inhibitory function of SFRs. The enhanced activation responses of SFR-deficient NK cells were dependent on integrin LFA-1 but not on DNAM-1 or NKG2D. SFR-mediated inhibition prevented the generation of activated forms of LFA-1. Hence, the Slam locus has an overall inhibitory role during NK cell activation that is solely dependent on 2B4. This effect is influenced by cytokines and leads to suppression of LFA-1 activity.