TCR signal strength controls thymic differentiation of discrete proinflammatory γδ T cell subsets.

The mouse thymus produces discrete γδ T cell subsets that make either interferon-γ (IFN-γ) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g(+/-) Cd3d(+/-) (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on γδ T cells. CD3DH mice had normal numbers and phenotypes of αß thymocyte subsets, but impaired differentiation of fetal Vγ6(+) (but not Vγ4(+)) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122(+) NK1.1(+) γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ(+) γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology.

Nonsense CD247 mutations show dominant-negative features in TCR expression and function.

BACKGROUND: The invariant TCRζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its three immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. OBJECTIVE: To analyze and model the potential impact on TCR expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. METHODS: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with wild-type CD247 or mutations ablating one (Q142X), two (Q101X), or three (Q70X) ITAMs. RESULTS: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2+3, respectively. Both mutations were associated with low surface CD3 expression, normal intracellular CD247 levels using a transmembrane-specific antibody but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO could not restore normal surface CD3 expression (only 60%, 22% and 10%, respectively), whereas in WT they reduced it (to 39%, 19% and 9% of normal levels), and both effects were ITAM number dependent. All six transfectants showed reduced CD69 induction (25-50%), indicating that they were unable to signal downstream properly neither isolated nor associated with wild-type CD247. CONCLUSION: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.

Inherited human ezrin deficiency impairs adaptive immunity.

BACKGROUND: Inborn errors of immunity (IEI) are a group of monogenic diseases that confer susceptibility to infection, autoimmunity, and cancer. Despite the life-threatening consequences of some IEI, their genetic cause remains unknown in many patients. OBJECTIVE: We investigated a patient with an IEI of unknown genetic etiology. METHODS: Whole-exome sequencing identified a homozygous missense mutation of the gene encoding ezrin (EZR), substituting a threonine for an alanine at position 129. RESULTS: Ezrin is one of the subunits of the ezrin, radixin, and moesin (ERM) complex. The ERM complex links the plasma membrane to the cytoskeleton and is crucial for the assembly of an efficient immune response. The A129T mutation abolishes basal phosphorylation and decreases calcium signaling, leading to complete loss of function. Consistent with the pleiotropic function of ezrin in myriad immune cells, multidimensional immunophenotyping by mass and flow cytometry revealed that in addition to hypogammaglobulinemia, the patient had low frequencies of switched memory B cells, CD4+ and CD8+ T cells, MAIT, γδ T cells, and centralnaive CD4+ cells. CONCLUSIONS: Autosomal-recessive human ezrin deficiency is a newly recognized genetic cause of B-cell deficiency affecting cellular and humoral immunity.

SLAM is a microbial sensor that regulates bacterial phagosome functions in macrophages.

Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.

Lymphocyte integration of complement cues.

We address current data, views and puzzles on the emerging topic of regulation of lymphocytes by complement proteins or fragments. Such regulation is believed to take place through complement receptors (CR) and membrane complement regulators (CReg) involved in cell function or protection, respectively, including intracellular signalling. Original observations in B cells clearly support that complement cues through CR improve their performance. Other lymphocytes likely integrate complement-derived signals, as most lymphoid cells constitutively express or regulate CR and CReg upon activation. CR-induced signals, particularly by anaphylatoxins, clearly regulate lymphoid cell function. In contrast, data obtained by CReg crosslinking using antibodies are not always confirmed in human congenital deficiencies or knock-out mice, casting doubts on their physiological relevance. Unsurprisingly, human and mouse complement systems are not completely homologous, adding further complexity to our still fragmentary understanding of complement-lymphocyte interactions.

Complement as a diagnostic tool in immunopathology.

The complement system is a complex and autoregulated multistep cascade at the interface of innate and adaptive immunity. It is activated by immune complexes or apoptotic cells (classical pathway), pathogen-associated glycoproteins (lectin pathway) or a variety of molecular and cellular surfaces (alternative pathway). Upon activation, complement triggers the generation of proteolytic fragments that allow the elimination of the activating surface by enhancing inflammation, opsonization, phagocytosis, and cellular lysis. Moreover, complement efficiently discriminates self from non-self surfaces by means of soluble and membrane-bound complement regulators which are critical for innate self-tolerance. Complement deficiency or dysfunction disturb complement homeostasis and give rise to diseases as diverse as bacterial infections, autoimmunity, or renal and neurological disorders. Research on complement-targeted therapies is an expanding field that has already improved the prognosis of severe diseases such as atypical Haemolytic Uremic syndrome or Paroxysmal Nocturnal Haemoglobinuria. Therefore, complement analysis and monitoring provides valuable information with deep implications for diagnosis and therapy. In addition to its important role as an extracellular defense system, it has now become evident that complement is also present intracellularly, and its activation has profound implications for leukocyte survival and function. In this review, we summarize the essential, up-to-date information on the use of complement as a diagnostic and therapeutic tool in the clinics.

Patients with CD3G mutations reveal a role for human CD3γ in Treg diversity and suppressive function.

Integrity of the T-cell receptor/CD3 complex is crucial for positive and negative selection of T cells in the thymus and for effector and regulatory functions of peripheral T lymphocytes. In humans, CD3D, CD3E, and CD3Z gene defects are a cause of severe immune deficiency and present early in life with increased susceptibility to infections. By contrast, CD3G mutations lead to milder phenotypes, mainly characterized by autoimmunity. However, the role of CD3γ in establishing and maintaining immune tolerance has not been elucidated. In this manuscript, we aimed to investigate abnormalities of T-cell repertoire and function in patients with genetic defects in CD3G associated with autoimmunity. High throughput sequencing was used to study composition and diversity of the T-cell receptor ß (TRB) repertoire in regulatory T cells (Tregs), conventional CD4+ (Tconv), and CD8+ T cells from 6 patients with CD3G mutations and healthy controls. Treg function was assessed by studying its ability to suppress proliferation of Tconv cells. Treg cells of patients with CD3G defects had reduced diversity, increased clonality, and reduced suppressive function. The TRB repertoire of Tconv cells from patients with CD3G deficiency was enriched for hydrophobic amino acids at positions 6 and 7 of the CDR3, a biomarker of self-reactivity. These data demonstrate that the T-cell repertoire of patients with CD3G mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition.

Analysis of the recovery of CD247 expression in a PID patient: insights into the spontaneous repair of defective genes.

Mutations in T-cell antigen receptor (TCR) subunit genes cause rare immunodeficiency diseases characterized by impaired expression of the TCR at the cell surface and selective T lymphopenia. Here, detailed analyses of spontaneously arising somatic mutations that recover CD247, and thus TCR expression, in a newly identified CD247-deficient patient are described. The recovery of CD247 expression in some patient T cells was associated with both reversion of the inactivating mutation and a variant with a compensating mutation that could reconstitute TCR expression, but not as efficiently as wild-type CD247. Multiple mutations were found in CD247 complementary DNAs (cDNAs) cloned from the patient as well as in cDNA and genomic DNA from other individuals, suggesting that genetic variation in this gene is frequent. Analyses of other genes mutated in primary immunodeficiency diseases (PIDs) where reversions have been described also revealed a higher rate of mutation than that observed for genes mutated in PIDs where revertants have not been identified or control genes. These data support the hypothesis that the occurrence of somatic mutations that may reconstitute genetic defects in PID is related to an increased propensity of those genes to mutate.

The Behçet's disease-associated variant of the aminopeptidase ERAP1 shapes a low-affinity HLA-B*51 peptidome by differential subpeptidome processing.

A low-activity variant of endoplasmic reticulum aminopeptidase 1 (ERAP1), Hap10, is associated with the autoinflammatory disorder Behçet's disease (BD) in epistasis with HLA-B*51, which is the main risk factor for this disorder. The role of Hap10 in BD pathogenesis is unknown. We sought to define the effects of Hap10 on the HLA-B*51 peptidome and to distinguish these effects from those due to HLA-B*51 polymorphisms unrelated to disease. The peptidome of the BD-associated HLA-B*51:08 subtype expressed in a Hap10-positive cell line was isolated, characterized by mass spectrometry, and compared with the HLA-B*51:01 peptidome from cells expressing more active ERAP1 allotypes. We additionally performed synthetic peptide digestions with recombinant ERAP1 variants and estimated peptide-binding affinity with standard algorithms. In the BD-associated ERAP1 context of B*51:08, longer peptides were generated; of the two major HLA-B*51 subpeptidomes with Pro-2 and Ala-2, the former one was significantly reduced, and the latter was increased and showed more ERAP1-susceptible N-terminal residues. These effects were readily explained by the low activity of Hap10 and the differential susceptibility of X-Pro and X-Ala bonds to ERAP1 trimming and together resulted in a significantly altered peptidome with lower affinity. The differences due to ERAP1 were clearly distinguished from those due to HLA-B*51 subtype polymorphism, which affected residue frequencies at internal positions of the peptide ligands. The alterations in the nature and affinity of HLA-B*51·peptide complexes probably affect T-cell and natural killer cell recognition, providing a sound basis for the joint association of ERAP1 and HLA-B*51 with BD.

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αß surface TCR expression.

BACKGROUND: The T cell antigen receptors (TCR) of αß and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αß or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αß and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/-) or CD3D (δ+/-, δ+/leaky) with that of normal controls. RESULTS: Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αß T lymphocytes in CD3γ+/- individuals, whereas CD3δ+/- and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αß TCR expression. CONCLUSIONS: The results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

MYC directly transactivates CR2/CD21, the receptor of the Epstein-Barr virus, enhancing the viral infection of Burkitt lymphoma cells.

MYC is an oncogenic transcription factor dysregulated in about half of total human tumors. While transcriptomic studies reveal more than 1000 genes regulated by MYC, a much smaller fraction of genes is directly transactivated by MYC. Virtually all Burkitt lymphoma (BL) carry chromosomal translocations involving MYC oncogene. Most endemic BL and a fraction of sporadic BL are associated with Epstein-Barr virus (EBV) infection. The currently accepted mechanism is that EBV is the BL-causing agent inducing MYC translocation. Herein we show that the EBV receptor, CR2 (also called CD21), is a direct MYC target gene. This is based on several pieces of evidence: MYC induces CR2 expression in both proliferating and arrested cells and in the absence of protein synthesis, binds the CR2 promoter and transactivates CR2 in an E-box-dependent manner. Moreover, using mice with conditional MYC ablation we show that MYC induces CR2 in primary B cells. Importantly, modulation of MYC levels directly correlates with EBV's ability of infection in BL cells. Altogether, in contrast to the widely accepted hypothesis for the correlation between EBV and BL, we propose an alternative hypothesis in which MYC dysregulation could be the first event leading to the subsequent EBV infection.

Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis.

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

New tools in regenerative medicine: gene therapy.

Gene therapy aims to transfer genetic material into cells to provide them with new functions. A gene transfer agent has to be safe, capable of expressing the desired gene for a sustained period of time in a sufficiently large population of cells to produce a biological effect. Identifying a gene transfer tool that meets all of these criteria has proven to be a difficult objective. Viral and nonviral vectors, in vivo, ex vivo and in situ strategies co-exist at present, although ex vivo lenti-or retroviral vectors are presently the most popular.Natural stem cells (from embryonic, hematopoietic, mesenchymal, or adult tissues) or induced progenitor stem (iPS) cells can be modified by gene therapy for use in regenerative medicine. Among them, hematopoietic stem cells have shown clear clinical benefit, but iPS cells hold humongous potential with no ethical concerns.

Hematopoietic stem cell transplantation for CD3δ deficiency.

BACKGROUND: CD3δ deficiency is a fatal form of severe combined immunodeficiency that can be cured by hematopoietic stem cell transplantation (HSCT). The presence of a thymus loaded with T-cell progenitors in patients with CD3δ deficiency may require special considerations in choosing the regimen of conditioning and the type of HSCT. OBJECTIVES: To study the outcome of CD3δ deficiency by using various modalities of stem cell transplantation. METHODS: We analyzed data on 13 patients with CD3δ deficiency who underwent HSCT in 7 centers. HSCT was performed by using different sources of donor stem cells as well as various conditioning regimens. RESULTS: One patient received stem cells from a matched related donor and survived after a second transplant, needing substantial conditioning in order to engraft. Only 2 of 7 other patients who received a mismatched related donor transplant survived; 2 of them had no conditioning, whereas the others received various combinations of conditioning regimens. Engraftment of T cells in the survivors appears incomplete. Three other patients who received stem cells from a matched unrelated donor survived and enjoyed full immune reconstitution. Two patients received unrelated cord blood without conditioning. One of them has had a partial but stable engraftment, whereas the other engrafted well but is only 12 months after HSCT. We also report here for the first time that patients with CD3δ deficiency can present with typical features of Omenn syndrome. CONCLUSIONS: HSCT is a successful treatment for patients with CD3δ deficiency. The small number of patients in this report prevents definitive statements on the importance of survival factors, but several are suggested: (1) HLA-matched donor transplants are associated with superior reconstitution and survival than are mismatched donor transplants; (2) substantial conditioning appears necessary; and (3) early diagnosis and absence of opportunistic infections may affect outcome.

Response to Vaccines in Patients with Immune-Mediated Inflammatory Diseases: A Narrative Review.

Patients with immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis and inflammatory bowel disease, are at increased risk of infection. International guidelines recommend vaccination to limit this risk of infection, although live attenuated vaccines are contraindicated once immunosuppressive therapy has begun. Biologic therapies used to treat IMIDs target the immune system to stop chronic pathogenic process but may also attenuate the protective immune response to vaccines. Here, we review the current knowledge regarding vaccine responses in IMID patients receiving treatment with biologic therapies, with a focus on the interleukin (IL)-12/23 inhibitors. B cell-depleting therapies, such as rituximab, strongly impair vaccines immunogenicity, and tumor necrosis factor (TNF) inhibitors and the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) fusion protein abatacept are also associated with attenuated antibody responses, which are further diminished in patients taking concomitant immunosuppressants. On the other hand, integrin, IL-6, IL-12/23, IL-17, and B-cell activating factor (BAFF) inhibitors do not appear to affect the immune response to several vaccines evaluated. Importantly, treatment with biologic therapies in IMID patients is not associated with an increased risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or developing severe disease. However, the efficacy of SARS-CoV-2 vaccines on IMID patients may be reduced compared with healthy individuals. The impact of biologic therapies on the response to SARS-CoV-2 vaccines seems to replicate what has been described for other vaccines. SARS-CoV-2 vaccination appears to be safe and well tolerated in IMID patients. Attenuated but, in general, still protective responses to SARS-CoV-2 vaccination in the context of certain therapies warrant current recommendations for a third primary dose in IMID patients treated with immunosuppressive drugs.