Microbiota stimulation generates LCMV-specific memory CD8+ T cells in SPF mice and determines their TCR repertoire during LCMV infection.

Both mouse and human harbour memory phenotype CD8+ T cells specific for antigens in hosts that have not been previously exposed to these antigens. The origin and the nature of the stimuli responsible for generation of CD44hi CD8+ T cells in specific pathogen-free (SPF) mice remain controversial. It is known that microbiota plays a crucial role in the prevention and resolution of systemic infections by influencing myelopoiesis, regulating dendritic cells, inflammasome activation and promoting the production of type I and II interferons. By contrast, here we suggest that microbiota has a direct effect on generation of memory phenotype CD44hiGP33+CD8+ T cells. In SPF mice, it generates a novel GP33+CD44hiCD8+ T cell sub-population associating the properties of innate and genuine memory cells. These cells are highly enriched in the bone marrow, proliferate rapidly and express immediate effector functions. They dominate the response to LCMV and express particular TCRß chains. The sequence of these selected TCRß chains overlaps with that of GP33+CD8+ T cells directly selected by microbiota in the gut epithelium of SPF mice, demonstrating a common selection mechanism in gut and peripheral CD8+ T cell pool. Therefore microbiota has a direct role in priming T cell immunity in SPF mice and in the selection of TCRß repertoires during systemic infection. We identify a mechanism that primes T cell immunity in SPF mice and may have a major role in colonization resistance and protection from infection.

Author Correction: B-cell depletion induces a shift in self antigen specific B-cell repertoire and cytokine pattern in patients with bullous pemphigoid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

B-cell depletion induces a shift in self antigen specific B-cell repertoire and cytokine pattern in patients with bullous pemphigoid.

Bullous Pemphigoid is the most common auto-immune bullous skin disease. It is characterized by the production of auto-antibodies directed against 2 proteins of the hemi-desmosome (BP180 and BP230). We assessed the efficacy and mechanisms of action of rituximab, an anti-CD20 monoclonal antibody, in 17 patients with severe and relapsing type of bullous pemphigoid. The phenotype, cytokine gene expression, and rearrangement of BP180-specific B-cell receptor genes were performed over 2 years following treatment. At the end of the study, 5 patients had died, 3 had withdrawn from the study, and 9 patients were in complete remission. The one- and two-year relapse rates were 44.1% (95% Confidence Interval (CI): 21.0-76.0%) and 66.5%, (95% CI: 38.4-91.4%), respectively. Phenotypic analyses confirmed dramatic B-cell depletion, which lasted for 9 to 12 months. The ELISA values of serum anti-BP180 antibodies and the frequency of BP180-specific circulating B cells decreased dramatically following treatment, which paralleled the improvement of skin lesions. During B-cell reconstitution, a polyclonal IgM repertoire appeared and a shift in the rearrangement of the B-cell receptor genes of BP180-specific circulating B cells was observed. Concurrently, we observed a decrease of IL-15, IL-6 and TNFα expressing BP180-specific B cells, and the emergence of IL-10 and IL-1RA-expressing BP180-specific IgM+ B cells in patients in complete remission off therapy, suggesting the functional plasticity of BP180-specific auto-immune B cells after rituximab treatment.

Respiratory Syncytial Virus Infects Regulatory B Cells in Human Neonates via Chemokine Receptor CX3CR1 and Promotes Lung Disease Severity.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in infants and is characterized by pulmonary infiltration of B cells in fatal cases. We analyzed the B cell compartment in human newborns and identified a population of neonatal regulatory B lymphocytes (nBreg cells) that produced interleukin 10 (IL-10) in response to RSV infection. The polyreactive B cell receptor of nBreg cells interacted with RSV protein F and induced upregulation of chemokine receptor CX3CR1. CX3CR1 interacted with RSV glycoprotein G, leading to nBreg cell infection and IL-10 production that dampened T helper 1 (Th1) cytokine production. In the respiratory tract of neonates with severe RSV-induced acute bronchiolitis, RSV-infected nBreg cell frequencies correlated with increased viral load and decreased blood memory Th1 cell frequencies. Thus, the frequency of nBreg cells is predictive of the severity of acute bronchiolitis disease and nBreg cell activity may constitute an early-life host response that favors microbial pathogenesis.

Public T cell receptors confer high-avidity CD4 responses to HIV controllers.

The rare patients who are able to spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants that underlie this response, we characterized the T cell receptor (TCR) repertoire directed at Gag293, the most immunoprevalent CD4 epitope in the HIV-1 capsid. HIV controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire that was characterized by a predominance of TRAV24 and TRBV2 variable genes, shared CDR3 motifs, and a high frequency of public clonotypes. The most prevalent public clonotypes generated TCRs with affinities at the higher end of values reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for the expression of these TCRs in HIV controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4+ T cells conferred high antigen sensitivity and polyfunctionality, thus recapitulating key features of the controller CD4 response. Transfer of a high-affinity Gag293-specific TCR also redirected CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. Together, these findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplification or transfer of such clonotypes may contribute to immunotherapeutic approaches aiming at a functional HIV cure.

Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients.

Chronic infection perturbs immune homeostasis. While prior studies have reported dysregulation of effector and memory cells, little is known about the effects on naïve T cell populations. We performed a cross-sectional study of chronic hepatitis C (cHCV) patients using tetramer-associated magnetic enrichment to study antigen-specific inexperienced CD8(+) T cells (i.e., tumor or unrelated virus-specific populations in tumor-free and sero-negative individuals). cHCV showed normal precursor frequencies, but increased proportions of memory-phenotype inexperienced cells, as compared to healthy donors or cured HCV patients. These observations could be explained by low surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated TCR hyperactivation and generation of potent CD8(+) T cell responses from the altered T cell repertoire of cHCV patients. In sum, we provide the first evidence that naïve CD8(+) T cells are dysregulated during cHCV infection, and establish a new mechanism of immune perturbation secondary to chronic infection.

Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation.

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.

An in vivo genetic reversion highlights the crucial role of Myb-Like, SWIRM, and MPN domains 1 (MYSM1) in human hematopoiesis and lymphocyte differentiation.

BACKGROUND: Myb-Like, SWIRM, and MPN domains 1 (MYSM1) is a metalloprotease that deubiquitinates the K119-monoubiquitinated form of histone 2A (H2A), a chromatin marker associated with gene transcription silencing. Likewise, it has been reported that murine Mysm1 participates in transcription derepression of genes, among which are transcription factors involved in hematopoietic stem cell homeostasis, hematopoiesis, and lymphocyte differentiation. However, whether MYSM1 has a similar function in human subjects remains unclear. Here we describe a patient presenting with a complete lack of B lymphocytes, T-cell lymphopenia, defective hematopoiesis, and developmental abnormalities. OBJECTIVES: We sought to characterize the underlying genetic cause of this syndrome. METHODS: We performed genome-wide homozygosity mapping, followed by whole-exome sequencing. RESULTS: Genetic analysis revealed that this novel disorder is caused by a homozygous MYSM1 missense mutation affecting the catalytic site within the deubiquitinase JAB1/MPN/Mov34 (JAMM)/MPN domain. Remarkably, during the course of our study, the patient recovered a normal immunohematologic phenotype. Genetic analysis indicated that this improvement originated from a spontaneous genetic reversion of the MYSM1 mutation in a hematopoietic stem cell. CONCLUSIONS: We here define a novel human immunodeficiency and provide evidence that MYSM1 is essential for proper immunohematopoietic development in human subjects. In addition, we describe one of the few examples of spontaneous in vivo genetic cure of a human immunodeficiency.

SYK expression endows human ZAP70-deficient CD8 T cells with residual TCR signaling.

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells.

Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome.

IMPORTANCE: Wiskott-Aldrich syndrome is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA antigen-matched allogeneic hematopoietic stem cell (HSC) transplantation is often curative but is associated with significant comorbidity. OBJECTIVE: To assess the outcomes and safety of autologous HSC gene therapy in Wiskott-Aldrich syndrome. DESIGN, SETTING, AND PARTICIPANTS: Gene-corrected autologous HSCs were infused in 7 consecutive patients with severe Wiskott-Aldrich syndrome lacking HLA antigen-matched related or unrelated HSC donors (age range, 0.8-15.5 years; mean, 7 years) following myeloablative conditioning. Patients were enrolled in France and England and treated between December 2010 and January 2014. Follow-up of patients in this intermediate analysis ranged from 9 to 42 months. INTERVENTION: A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector. MAIN OUTCOMES AND MEASURES: Primary outcomes were improvement at 24 months in eczema, frequency and severity of infections, bleeding tendency, and autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and hematological characteristics and evidence of safety through vector integration analysis. RESULTS: Six of the 7 patients were alive at the time of last follow-up (mean and median follow-up, 28 months and 27 months, respectively) and showed sustained clinical benefit. One patient died 7 months after treatment of preexisting drug-resistant herpes virus infection. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5 of 5 patients. No severe bleeding episodes were recorded after treatment, and at last follow-up, all 6 surviving patients were free of blood product support and thrombopoietic agonists. Hospitalization days were reduced from a median of 25 days during the 2 years before treatment to a median of 0 days during the 2 years after treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis. CONCLUSIONS AND RELEVANCE: This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcomes and safety.

PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity.

BACKGROUND: PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. OBJECTIVE: We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. METHODS: Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. RESULTS: We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. CONCLUSION: Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.

A modified γ-retrovirus vector for X-linked severe combined immunodeficiency.

BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).

CD4 T cells with effector memory phenotype and function develop in the sterile environment of the fetus.

The T cell compartment is considered to be naïve and dedicated to the development of tolerance during fetal development. We have identified and characterized a population of fetally developed CD4 T cells with an effector memory phenotype (TEM), which are present in cord blood. This population is polyclonal and has phenotypic features similar to those of conventional adult memory T cells, such as CD45RO expression. These cells express low levels of CD25 but are distinct from regulatory T cells because they lack Foxp3 expression. After T cell receptor activation, neonatal TEM cells readily produced tumor necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We also detected interferon-γ (IFN-γ)-producing T helper 1 (TH1) cells and interleukin-4 (IL-4)/IL-13-producing TH2-like cells, but not IL-17-producing cells. We used chemokine receptor expression patterns to divide this TEM population into different subsets and identified distinct transcriptional programs using whole-genome microarray analysis. IFN-γ was found in CXCR3(+) TEM cells, whereas IL-4 was found in both CXCR3(+) TEM cells and CCR4(+) TEM cells. CCR6(+) TEM cells displayed a genetic signature that corresponded to TH17 cells but failed to produce IL-17A. However, the TH17 function of TEM cells was observed in the presence of IL-1ß and IL-23. In summary, in the absence of reported pathology or any major infectious history, T cells with a memory-like phenotype develop in an environment thought to be sterile during fetal development and display a large variety of inflammatory effector functions associated with CD4 TH cells at birth.

A recurrent dominant negative E47 mutation causes agammaglobulinemia and BCR(-) B cells.

Approximately 90% of patients with isolated agammaglobulinemia and failure of B cell development have mutations in genes required for signaling through the pre­B cell and B cell receptors. The nature of the gene defect in the majority of remaining patients is unknown. We recently identified 4 patients with agammaglobulinemia and markedly decreased numbers of peripheral B cells. The B cells that could be detected had an unusual phenotype characterized by the increased expression of CD19 but the absence of a B cell receptor. Genetic studies demonstrated that all 4 patients had the exact same de novo mutation in the broadly expressed transcription factor E47. The mutant protein (E555K) was stable in patient-derived EBV-transformed cell lines and cell lines transfected with expression vectors. E555K in the transfected cells localized normally to the nucleus and resulted in a dominant negative effect when bound to DNA as a homodimer with wild-type E47. Mutant E47 did permit DNA binding by a tissue-specific heterodimeric DNA-binding partner, myogenic differentiation 1 (MYOD). These findings document a mutational hot-spot in E47 and represent an autosomal dominant form of agammaglobulinemia. Further, they indicate that E47 plays a critical role in enforcing the block in development of B cell precursors that lack functional antigen receptors.

HLA-A*01:03, HLA-A*24:02, HLA-B*08:01, HLA-B*27:05, HLA-B*35:01, HLA-B*44:02, and HLA-C*07:01 monochain transgenic/H-2 class I null mice: novel versatile preclinical models of human T cell responses.

We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human ß2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8(+) T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ-producing CD8(+) T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8(+) T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell-based vaccines.

Long-term remissions of severe pemphigus after rituximab therapy are associated with prolonged failure of desmoglein B cell response.

Pemphigus is a severe blistering condition of the skin and mucosa caused by autoantibodies directed against desmogleins, which are a type of keratinocyte adhesion protein. B cell depletion by rituximab has short-term efficacy against pemphigus. We aimed to assess the long-term course of pemphigus patients after B cell depletion and to understand the immunological mechanisms that mediate long-lasting remissions. We evaluated the clinical course of 22 pemphigus patients treated with rituximab after a 79-month median follow-up and compared the anti-desmoglein B cell response and B and T lymphocyte subpopulations and repertoire between patients who achieved complete remission (CR) and those who had incomplete remission (IR). Thirteen patients (59%) experienced CR during the study, including 10 patients off treatment and 3 patients with prednisone doses <10 mg/day; 9 patients had IR. A marked increase was observed in the ratio of CD19(+)CD27(-) naïve B cells to CD19(+)CD27(+) memory B cells. Indeed, patients in CR had a fourfold higher number of transitional B cells and interleukin-10-secreting regulatory B cells than those in IR. Furthermore, CR was associated with modification of the initial B cell repertoire and the disappearance of desmoglein-specific circulating immunoglobulin G-positive (IgG(+)) B lymphocytes, whereas a skewed B cell repertoire was observed in patients in IR. Thus, a blockage of B cell maturation, a prolonged repopulation with naïve B cells, and a delayed reappearance of memory B cells, which resulted in the disappearance of circulating desmoglein-specific IgG(+) B lymphocytes, contribute to the long-lasting effectiveness of rituximab for treating pemphigus.

Whole-exome sequencing identifies Coronin-1A deficiency in 3 siblings with immunodeficiency and EBV-associated B-cell lymphoproliferation.

BACKGROUND: Primary immunodeficiencies are a rare group of inborn diseases characterized by a broad clinical and genetic heterogeneity. Substantial advances in the identification of the underlying molecular mechanisms can be achieved through the study of patients with increased susceptibility to specific infections and immune dysregulation. We evaluated 3 siblings from a consanguineous family presenting with EBV-associated B-cell lymphoproliferation at an early age (12, 7½, and 14 months, respectively) and profound naive T-cell lymphopenia. OBJECTIVE: On the basis of the hypothesis of a rare inborn immunodeficiency of autosomal recessive inheritance, we sought to characterize the underlying genetic defect. METHODS: We performed genome-wide homozygosity mapping, followed by whole-exome sequencing. RESULTS: We identified a homozygous inherited missense mutation in the gene encoding Coronin-1A (CORO1A) in the 3 siblings. This mutation, p. V134M, results in the substitution of an evolutionarily conserved amino acid within the ß-propeller domain, which abrogates almost completely the protein expression in the patients' cells. In addition to a significant diminution of naive T-cell numbers, we found impaired development of a diverse T-cell repertoire, near-to-absent invariant natural killer T cells, and severely diminished mucosal-associated invariant T cell numbers. CONCLUSIONS: Our findings define a new clinical entity of a primary immunodeficiency with increased susceptibility to EBV-induced lymphoproliferation in patients associated with hypomorphic Coronin-1A mutation.

EVER2 deficiency is associated with mild T-cell abnormalities.

Epidermodysplasia verruciformis (EV) is a rare genodermatosis characterized by persistent flat warts or pityriasis versicolor-like lesions caused by betapapillomaviruses (EV-HPVs). Autosomal recessive EVER1 and EVER2 deficiencies account for EV in most patients. The mechanisms by which mutations in these partners of the Zinc transporter ZnT1 impair host defense against EV-HPVs are still poorly understood. Keratinocytes of EVER-deficient patients display an alteration of zinc homeostasis and an enhanced proliferative activity. Since EVER proteins are highly expressed in T lymphocytes, we aimed to assess the impact of EVER2 deficiency on T-cell development and function. We studied circulating lymphocyte populations in three adult EV patients sharing the same EVER2 mutation (T150fsX3). We found a normal count of CD4(+) and CD8(+) T cells and a normal proliferative capacity in response to anti-CD3 stimulation. However, we observed a significant increase of memory CD4(+) and effector memory CD8(+) T cells, a bias of the TCR Vαß and Vγδ repertoires and an increase of skin-homing CD4(+) T-cell subsets. Our findings suggest that EVER2-deficient patients display mild T-cell abnormalities. It remains unclear whether these abnormalities result from EVER deficiency, chronic EV-HPV infection, or both.

Cernunnos deficiency reduces thymocyte life span and alters the T cell repertoire in mice and humans.

Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice.

Polymerase ε1 mutation in a human syndrome with facial dysmorphism, immunodeficiency, livedo, and short stature ("FILS syndrome").

DNA polymerase ε (Polε) is a large, four-subunit polymerase that is conserved throughout the eukaryotes. Its primary function is to synthesize DNA at the leading strand during replication. It is also involved in a wide variety of fundamental cellular processes, including cell cycle progression and DNA repair/recombination. Here, we report that a homozygous single base pair substitution in POLE1 (polymerase ε 1), encoding the catalytic subunit of Polε, caused facial dysmorphism, immunodeficiency, livedo, and short stature ("FILS syndrome") in a large, consanguineous family. The mutation resulted in alternative splicing in the conserved region of intron 34, which strongly decreased protein expression of Polε1 and also to a lesser extent the Polε2 subunit. We observed impairment in proliferation and G1- to S-phase progression in patients' T lymphocytes. Polε1 depletion also impaired G1- to S-phase progression in B lymphocytes, chondrocytes, and osteoblasts. Our results evidence the developmental impact of a Polε catalytic subunit deficiency in humans and its causal relationship with a newly recognized, inherited disorder.