CD5L as a promising biological therapeutic for treating sepsis.

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L's important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L-/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans.

Loss of the KN Motif and AnKyrin Repeat Domain 1 (KANK1) Leads to Lymphoid Compartment Dysregulation in Murine Model.

The KN Motif and AnKyrin Repeat Domain 1 (KANK1) is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the KANK1 gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for Kank1 in lymphoid cell development, and total protein analysis suggests the involvement of Kank1 in BM cells' cytoskeleton formation and mobility.

Anti-HVEM mAb therapy improves antitumoral immunity both in vitro and in vivo, in a novel transgenic mouse model expressing human HVEM and BTLA molecules challenged with HVEM expressing tumors.

BACKGROUND: Tumor necrosis factor superfamily member 14 (TNFRSF14)/herpes virus entry mediator (HVEM) is the ligand for B and T lymphocyte attenuator (BTLA) and CD160-negative immune co-signaling molecules as well as viral proteins. Its expression is dysregulated with an overexpression in tumors and a connection with tumors of adverse prognosis. METHODS: We developed C57BL/6 mouse models co-expressing human (hu)BTLA and huHVEM as well as antagonistic monoclonal antibodies (mAbs) that completely prevent the interactions of HVEM with its ligands. RESULTS: Here, we show that the anti-HVEM18-10 mAb increases primary human αß-T cells activity alone (CIS-activity) or in the presence of HVEM-expressing lung or colorectal cancer cells in vitro (TRANS-activity). Anti-HVEM18-10 synergizes with antiprogrammed death-ligand 1 (anti-PD-L1) mAb to activate T cells in the presence of PD-L1-positive tumors, but is sufficient to trigger T cell activation in the presence of PD-L1-negative cells. In order to better understand HVEM18-10 effects in vivo and especially disentangle its CIS and TRANS effects, we developed a knockin (KI) mouse model expressing human BTLA (huBTLA+/+) and a KI mouse model expressing both huBTLA+/+/huHVEM+/+ (double KI (DKI)). In vivo preclinical experiments performed in both mouse models showed that HVEM18-10 treatment was efficient to decrease human HVEM+ tumor growth. In the DKI model, anti-HVEM18-10 treatment induces a decrease of exhausted CD8+ T cells and regulatory T cells and an increase of effector memory CD4+ T cells within the tumor. Interestingly, mice which completely rejected tumors (±20%) did not develop tumors on rechallenge in both settings, therefore showing a marked T cell-memory phenotype effect. CONCLUSIONS: Altogether, our preclinical models validate anti-HVEM18-10 as a promising therapeutic antibody to use in clinics as a monotherapy or in combination with existing immunotherapies (antiprogrammed cell death protein 1/anti-PD-L1/anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4)).

A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

High-throughput mass cytometry staining for deep phenotyping of human natural killer cells.

This protocol details the step-by-step procedure for in-depth immune phenotyping of peripheral blood natural killer (NK) cells from clinical samples by mass cytometry. The protocol consists of three main steps: PBMC incubation with a mix of metal-conjugated antibodies for extracellular phenotyping followed by fixation, permeabilization and incubation with a mix of metal-conjugated antibodies for staining of intracellular proteins, and sample acquisition on a mass cytometer. High-dimensional analysis enables the visualization of NK cell subsets and their phenotypical characteristics. For complete details on the use and execution of this protocol, please refer to Chretien et al. (2021).

Single-cell transcriptomics uncovers an instructive T-cell receptor role in adult γδ T-cell lineage commitment.

After entering the adult thymus, bipotent T-cell progenitors give rise to αß or γδ T cells. To determine whether the γδ T-cell receptor (TCR) has an instructive role in γδ T-cell lineage commitment or only "confirms" a pre-established γδ Τ-cell lineage state, we exploited mice lacking expression of LAT, an adaptor required for γδ TCR signaling. Although these mice showed a T-cell development block at the CD4- CD8- double-negative third (DN3) stage, 0.3% of their DN3 cells expressed intermediate levels of γδ TCR (further referred to as γδint ) at their surface. Single-cell transcriptomics of LAT-deficient DN3 γδint cells demonstrated no sign of commitment to the γδ T-cell lineage, apart from γδ TCR expression. Although the lack of LAT is thought to tightly block DN3 cell development, we unexpectedly found that 25% of LAT-deficient DN3 γδint cells were actively proliferating and progressed up to the DN4 stage. However, even those cells failed to turn on the transcriptional program associated with the γδ T-cell lineage. Therefore, the γδ TCR-LAT signaling axis builds upon a γδ T-cell uncommitted lineage state to fully instruct adult γδ T-cell lineage specification.

Conditional Deletion of Pdcd1 Identifies the Cell-Intrinsic Action of PD-1 on Functional CD8 T Cell Subsets for Antitumor Efficacy.

Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity.

High-dimensional mass cytometry analysis of NK cell alterations in AML identifies a subgroup with adverse clinical outcome.

Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56-CD16+ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56-CD16+ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56-CD16+ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56-CD16+ NK cells. Overall, our data suggest that the accumulation of CD56-CD16+ NK cells may be the consequence of immune escape from innate immunity during AML progression.

LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN2 as a regulator of effector functions.

T-cell receptor (TCR) ligation-mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time-resolved high-resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T-cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast-track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T-cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T-cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.

Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4+ T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.

In Utero Administration of Drugs Targeting Microglia Improves the Neurodevelopmental Outcome Following Cytomegalovirus Infection of the Rat Fetal Brain.

Congenital cytomegalovirus (CMV) infections represent one leading cause of neurodevelopmental disorders. Recently, we reported on a rat model of CMV infection of the developing brain in utero, characterized by early and prominent infection and alteration of microglia-the brain-resident mononuclear phagocytes. Besides their canonical function against pathogens, microglia are also pivotal to brain development. Here we show that CMV infection of the rat fetal brain recapitulated key postnatal phenotypes of human congenital CMV including increased mortality, sensorimotor impairment reminiscent of cerebral palsy, hearing defects, and epileptic seizures. The possible influence of early microglia alteration on those phenotypes was then questioned by pharmacological targeting of microglia during pregnancy. One single administration of clodronate liposomes in the embryonic brains at the time of CMV injection to deplete microglia, and maternal feeding with doxycyxline throughout pregnancy to modify microglia in the litters' brains, were both associated with dramatic improvements of survival, body weight gain, sensorimotor development and with decreased risk of epileptic seizures. Improvement of microglia activation status did not persist postnatally after doxycycline discontinuation; also, active brain infection remained unchanged by doxycycline. Altogether our data indicate that early microglia alteration, rather than brain CMV load per se, is instrumental in influencing survival and the neurological outcomes of CMV-infected rats, and suggest that microglia might participate in the neurological outcome of congenital CMV in humans. Furthermore this study represents a first proof-of-principle for the design of microglia-targeted preventive strategies in the context of congenital CMV infection of the brain.

Diversity of innate immune cell subsets across spatial and temporal scales in an EAE mouse model.

In both multiple sclerosis and its model experimental autoimmune encephalomyelitis (EAE), the extent of resident microglia activation and infiltration of monocyte-derived cells to the CNS is positively correlated to tissue damage. To address the phenotype characterization of different cell subsets, their spatio-temporal distributions and contributions to disease development we induced EAE in Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined high content flow cytometry, immunofluorescence and two-photon imaging in live mice and identified a stepwise program of inflammatory cells accumulation. First on day 10 after induction, EGFP+ neutrophils and monocytes invade the spinal cord parenchyma through the meninges rather than by extravasion. This event occurs just before axonal losses in the white matter. Once in the parenchyma, monocytes mature into EGFP+/EYFP+ monocyte-derived dendritic cells (moDCs) whose density is maximal on day 17 when the axonal degradation and clinical signs stabilize. Meanwhile, microglia is progressively activated in the grey matter and subsequently recruited to plaques to phagocyte axon debris. LysM-EGFP//CD11c-EYFP mice appear as a powerful tool to differentiate moDCs from macrophages and to study the dynamics of immune cell maturation and phenotypic evolution in EAE.

Atp6ap2 ablation in adult mice impairs viability through multiple organ deficiencies.

ATP6AP2 codes for the (pro)renin receptor and is an essential component of vacuolar H+ ATPase. Activating (pro)renin for conversion of Angiotensinogen to Angiotensin makes ATP6AP2 attractive for drug intervention. Tissue-specific ATP6AP2 inactivation in mouse suggested a strong impact on various organs. Consistent with this, we found that embryonic ablation of Atp6ap2 resulted in both male hemizygous lethality and female haploinsufficiency. Next, we examined the phenotype of an induced inactivation in the adult animal, most akin to detect potential effect of functional interference of ATP6AP2 through drug therapy. Induced ablation of Atp6ap2, even without equal efficiency in all tissues (aorta, brain and kidney), resulted in rapid lethality marked by weight loss, changes in nutritional as well as blood parameters, leukocyte depletion, and bone marrow hypoplasia. Upon Atp6ap2 ablation, the colon demonstrated a rapid disruption of crypt morphology, aberrant proliferation, cell-death activation, as well as generation of microadenomas. Consequently, disruption of ATP6AP2 is extremely poorly tolerated in the adult, and severely affects various organ systems demonstrating that ATP6AP2 is an essential gene implicated in basic cellular mechanisms and necessary for multiple organ function. Accordingly, any potential drug targeting of this gene product must be strictly assessed for safety.

Natural Killer Defective Maturation Is Associated with Adverse Clinical Outcome in Patients with Acute Myeloid Leukemia.

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients (N = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) (P = 0.0006) and relapse-free survival (RFS) (P < 0.0001) were observed among these different groups. Patients with hypomaturation profile had reduced OS, with 3-year OS rates of 12.5 vs 57.1 and 57.4% for patients with intermediate and hypermaturation, respectively. Consistently, patients with hypomaturation profile had reduced RFS, with 3-year RFS rates of 0 vs 52.6 and 73.3% for patients with intermediate and hypermaturation, respectively. In multivariate Cox regression models, NK hypomaturation remained significantly associated with reduced OS and RFS, independent of other factors [hazard ratio (HR) = 4.15, P = 0.004 and HR = 8.23, P = 0.003, respectively]. NK maturation defects were further explored by mass cytometry and revealed that NK hypomaturation profile is associated with a reduced frequency of memory-like NK cells. In conclusion, besides classical alterations of NK triggering and inhibitory receptors expression in AML, we confirm that the homeostasis of NK maturation can be modified in the context of AML, notably with a deep maturation blockade in almost 10% patients.

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species.

Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.

Broad and Largely Concordant Molecular Changes Characterize Tolerogenic and Immunogenic Dendritic Cell Maturation in Thymus and Periphery.

Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.

Cytomegalovirus Infection of the Rat Developing Brain In Utero Prominently Targets Immune Cells and Promotes Early Microglial Activation.

BACKGROUND: Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells. OBJECTIVES AND METHODS: In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments. RESULTS: Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b- lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1. CONCLUSION: In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental, those putative double-edge swords events actually play.

Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.

Inflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP(+) cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP(+) cells. Fluorescent invading cells were conventional XCR1(+) and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP(+)/EYFP(+) monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response.

Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.

The recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five-fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry-based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms.

Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3⁺ and Foxp3⁻ T cells.

Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.