ABSTRACT
The expression levels of TCRs on the surface of human T cells define the avidity of TCR-HLA/peptide interactions. In this study, we have explored which components of the TCR-CD3 complex are involved in determining the surface expression levels of TCRs in primary human T cells. The results show that there is a surplus of endogenous TCR α/ß chains that can be mobilised by providing T cells with additional CD3γ,δ,ε,ζ chains, which leads to a 5-fold increase in TCR α/ß surface expression. The analysis of individual CD3 chains revealed that provision of additional ζ chain alone was sufficient to achieve a 3-fold increase in endogenous TCR expression. Similarly, CD3ζ also limits the expression levels of exogenous TCRs transduced into primary human T cells. Interestingly, transduction with TCR plus CD3ζ not only increased surface expression of the introduced TCR, but it also reduced mispairing with endogenous TCR chains, resulting in improved antigen-specific function. TCR reconstitution experiments in HEK293T cells that do not express endogenous TCR or CD3 showed that TCRα/ß and all four CD3 chains were required for optimal surface expression, while in the absence of CD3ζ the TCR expression was reduced by 50%. Together, the data show that CD3ζ is a key regulator of TCR expression levels in human T cells, and that gene transfer of exogenous TCR plus CD3ζ improved TCR surface expression, reduced TCR mispairing and increased antigen-specific function.
Subject(s)
CD3 Complex , Humans , CD3 Complex/immunology , CD3 Complex/metabolism , CD3 Complex/genetics , HEK293 Cells , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/metabolism , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Receptors, Antigen, T-Cell, alpha-beta/immunology , Lymphocyte Activation/immunology , Receptor-CD3 Complex, Antigen, T-Cell/immunology , Receptor-CD3 Complex, Antigen, T-Cell/metabolism , Receptor-CD3 Complex, Antigen, T-Cell/geneticsABSTRACT
Multiple sclerosis is an autoinflammatory condition that results in damage to myelinated neurons in affected patients. While disease-modifying treatments have been successful in slowing the progression of relapsing-remitting disease, most patients still progress to secondary progressive disease that is largely unresponsive to disease-modifying treatments. Similarly, there is currently no effective treatment for patients with primary progressive MS. Innate and adaptive immune cells in the CNS play a critical role in initiating an autoimmune attack and in maintaining the chronic inflammation that drives disease progression. In this review, we will focus on recent insights into the role of T cells with regulatory function in suppressing the progression of MS, and, more importantly, in promoting the remyelination and repair of MS lesions in the CNS. We will discuss the exciting potential to genetically reprogram regulatory T cells to achieve immune suppression and enhance repair locally at sites of tissue damage, while retaining a fully competent immune system outside the CNS. In the future, reprogramed regulatory T cells with defined specificity and function may provide life medicines that can persist in patients and achieve lasting disease suppression after one cycle of treatment.
Subject(s)
Multiple Sclerosis , T-Lymphocytes, Regulatory , Humans , T-Lymphocytes, Regulatory/immunology , Multiple Sclerosis/immunology , Multiple Sclerosis/drug therapy , Multiple Sclerosis/therapy , Animals , Antigens/immunology , Molecular Targeted TherapyABSTRACT
The therapeutic success and widespread approval of genetically engineered T cells for a variety of hematologic malignancies spurred the development of synthetic cell-based immunotherapies for CNS lymphoma, primary brain tumors, and a growing spectrum of nononcologic disease conditions of the nervous system. Chimeric antigen receptor effector T cells bear the potential to deplete target cells with higher efficacy, better tissue penetration, and greater depth than antibody-based cell depletion therapies. In multiple sclerosis and other autoimmune disorders, engineered T-cell therapies are being designed and currently tested in clinical trials for their safety and efficacy to eliminate pathogenic B-lineage cells. Chimeric autoantibody receptor T cells expressing a disease-relevant autoantigen as cell surface domains are designed to selectively deplete autoreactive B cells. Alternative to cell depletion, synthetic antigen-specific regulatory T cells can be engineered to locally restrain inflammation, support immune tolerance, or efficiently deliver neuroprotective factors in brain diseases in which current therapeutic options are very limited. In this article, we illustrate prospects and bottlenecks for the clinical development and implementation of engineered cellular immunotherapies in neurologic diseases.
Subject(s)
Artificial Cells , Brain Diseases , Nervous System Diseases , Humans , Nervous System Diseases/therapy , Immunotherapy , AutoantibodiesABSTRACT
T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable ß domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.
Subject(s)
Receptors, Antigen, T-Cell , T-Lymphocytes , Humans , Antigens/metabolism , Genes, T-Cell Receptor , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Complementarity Determining RegionsABSTRACT
Regulatory T cells (Treg) are potent inhibitors of autoreactive T cells. The intracellular transcription factor FoxP3 controls the expression levels of a diverse set of genes and plays a critical role in programming functional Tregs. Although, antigen-specific Tregs are more potent than polyclonal Tregs in treating ongoing autoimmunity, phenotype plasticity associated with loss of FoxP3 expression in Tregs can lead to the conversion into antigen-specific effector T cells which might exacerbate autoimmune pathology. In this study, we designed a retroviral vector driving the expression of FoxP3 and a human HLA-DR-restricted TCR from the same promoter. Transduction of purified human Tregs revealed that all TCR-positive cells had elevated levels of FoxP3 expression, increased CD25 and CTLA4 expression and potent suppressive function. Elevated FoxP3 expression did not impair the in vitro expansion of engineered Tregs. Adoptive transfer into HLA-DR transgenic mice revealed that FoxP3+TCR engineered Tregs showed long-term persistence with stable FoxP3 and TCR expression. In contrast, adoptive transfer of Tregs engineered with TCR only resulted in the accumulation of TCR-positive, FoxP3-negative T cells which displayed antigen-specific effector function when stimulated with the TCR-recognised peptides. Our data indicate that forced expression of FoxP3 can prevent accumulation of antigen-specific effector T cells without impairing the engraftment and persistence of engineered Tregs.
Subject(s)
Autoimmunity , T-Lymphocytes, Regulatory , Humans , Animals , Mice , Adoptive Transfer , Mice, Transgenic , Forkhead Transcription Factors/genetics , Receptors, Antigen, T-Cell/geneticsABSTRACT
NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.
Subject(s)
HLA-B7 Antigen/immunology , Immunodominant Epitopes/immunology , Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , T-Lymphocytes, Cytotoxic/immunology , Aged , Amino Acid Sequence , Antibodies, Viral/immunology , Antibody Affinity/immunology , COVID-19/immunology , COVID-19/pathology , Cell Line, Transformed , Female , Gene Expression Profiling , Humans , Immunologic Memory/immunology , Male , Middle Aged , Receptors, Antigen, T-Cell/immunology , Severity of Illness Index , Vaccinia virus/genetics , Vaccinia virus/immunology , Vaccinia virus/metabolismABSTRACT
Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.
Subject(s)
Enzyme Inhibitors/pharmacology , Hepatitis B virus/drug effects , Sterol O-Acyltransferase/antagonists & inhibitors , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/virology , Drug Therapy, Combination , Enzyme Inhibitors/administration & dosage , Hepatitis B virus/immunology , Hepatitis B virus/pathogenicity , Hepatitis B, Chronic/drug therapy , Humans , Immune Checkpoint Inhibitors/administration & dosage , Immune Checkpoint Inhibitors/pharmacology , In Vitro Techniques , Liver/drug effects , Liver/immunology , Liver/virology , Liver Neoplasms/drug therapy , Liver Neoplasms/virology , T-Lymphocytes/immunologyABSTRACT
Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines show protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, spike. Because new SARS-CoV-2 variants are emerging rapidly, as exemplified by the B.1.1.7, B.1.351, and P.1 lineages, it is critical to understand whether antibody responses induced by infection with the original SARS-CoV-2 virus or current vaccines remain effective. In this study, we evaluate neutralization of a series of mutated spike pseudotypes based on divergence from SARS-CoV and then compare neutralization of the B.1.1.7 spike pseudotype and individual mutations. Spike-specific monoclonal antibody neutralization is reduced dramatically; in contrast, polyclonal antibodies from individuals infected in early 2020 remain active against most mutated spike pseudotypes, but potency is reduced in a minority of samples. This work highlights that changes in SARS-CoV-2 spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their effect on vaccine efficacy.
Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Antibody Formation , COVID-19/immunology , COVID-19/metabolism , COVID-19 Vaccines/immunology , HEK293 Cells , Humans , Neutralization Tests/methods , Point Mutation , Receptors, Virus/genetics , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
Adoptive immunotherapy with gene-engineered T cells has provided new treatment options for cancer patients [...].
Subject(s)
Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Genetic Therapy/methods , Humans , Immunotherapy, Adoptive/methods , Neoplasms/immunology , Receptors, Chimeric Antigen/immunology , Tumor Microenvironment/immunologyABSTRACT
The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases.
Subject(s)
Stromal Cells , Thymus Gland/immunology , Animals , Autoimmunity , Cell Differentiation , Epithelial Cells/immunology , Extracellular Matrix , Female , Humans , Male , Mice , Mice, Nude , Rats , Regeneration , Thymocytes , Thymus Gland/pathology , Thymus Gland/transplantation , Tissue ScaffoldsABSTRACT
This review presents key advances in combining T cell receptor (TCR) gene transfer to redirect T-cell specificity with gene engineering in order to enhance cancer-protective immune function. We discuss how emerging insights might be applied to CD4+ T cells. Although much attention has been paid to the role of CD8+ cytotoxic T cells in tumour protection, we provide convincing evidence that CD4+ helper T cells play a critical role in cancer immune responses in animal models and also in patients. We demonstrate that genetic engineering technologies provide exciting opportunities to extend the specificity range of CD4+ T cells from MHC class-II-presented epitopes to include peptides presented by MHC class I molecules. Functional enhancement of tumour immunity can improve the sensitivity of T cells to cancer antigens, promote survival in a hostile tumour microenvironment, boost cancer-protective effector mechanisms and enable the formation of T-cell memory. Engineered cancer-specific CD4+ T cells may contribute to protective immunity by a direct pathway involving cancer cell killing, and by an indirect pathway that boosts the function, persistence and memory formation of CD8+ T cells.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Engineering/methods , Neoplasms/immunology , Animals , Cytokines/metabolism , Humans , Immunologic Memory , Signal TransductionABSTRACT
Acute graft-versus-host disease (GVHD) is initially triggered by alloreactive T cells, which damage peripheral tissues and lymphoid organs. Subsequent transition to chronic GVHD involves the emergence of autoimmunity, although the underlying mechanisms driving this process are unclear. Here, we tested the hypothesis that acute GVHD blocks peripheral tolerance of autoreactive T cells by impairing lymph node (LN) display of peripheral tissue-restricted antigens (PTAs). At the initiation of GVHD, LN fibroblastic reticular cells (FRCs) rapidly reduced expression of genes regulated by DEAF1, an autoimmune regulator-like transcription factor required for intranodal expression of PTAs. Subsequently, GVHD led to the selective elimination of the FRC population, and blocked the repair pathways required for its regeneration. We used a transgenic mouse model to show that the loss of presentation of an intestinal PTA by FRCs during GVHD resulted in the activation of autoaggressive T cells and gut injury. Finally, we show that FRCs normally expressed a unique PTA gene signature that was highly enriched for genes expressed in the target organs affected by chronic GVHD. In conclusion, acute GVHD damages and prevents repair of the FRC network, thus disabling an essential platform for purging autoreactive T cells from the repertoire.
Subject(s)
Autoantigens/immunology , Autoimmunity , Graft vs Host Disease/immunology , Intestinal Diseases/immunology , Lymph Nodes/immunology , T-Lymphocytes/immunology , Animals , Autoantigens/genetics , Graft vs Host Disease/genetics , Graft vs Host Disease/pathology , Intestinal Diseases/genetics , Intestinal Diseases/pathology , Lymph Nodes/pathology , Mice , Mice, Knockout , T-Lymphocytes/pathologyABSTRACT
TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans.
Subject(s)
Antigens/immunology , Cell Engineering , Genes, T-Cell Receptor/genetics , Genes, T-Cell Receptor/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Animals , Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Cell Line, Tumor , Cell Proliferation , Cytokines/metabolism , Gene Expression , Genetic Therapy , Humans , Lectins, C-Type/metabolism , Lymphocyte Activation , Male , Mice , Mice, Inbred NOD , Mice, SCID , Models, Molecular , Protein Domains , Protein Engineering , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunologyABSTRACT
We explored whether engineering of T cell specificity and effector function improves immunotherapy of solid tumors. Although IL-12 can enhance cancer immunity, a strategy of safe IL-12 delivery without toxicity is currently lacking. We engineered T cells to express IL-12 controlled by the NFAT promoter responsive to TCR stimulation, or by the Tet-On promoter responsive to doxycycline. In vivo, NFAT-engineered T cells caused lethal toxicity, while Tet-engineered T cells were safe in the absence of doxycycline. Combining gene transfer of the melanoma-specific TRP2-TCR with Tet-IL-12 engineering revealed that temporal induction of IL-12 was essential to inhibit the growth of B16F10 melanoma tumors. Induced IL-12 increased the number of tumor-infiltrating T cells and also prevented the down-modulation of the TRP2-TCR and the associated up-regulation of the PD1 marker that was observed in the absence of IL-12. In addition, temporal induction of IL-12 expression also increased the number of plasmacytoid DC in the tumor micro-environment. We show that repeated induction of IL-12 can be used to enhance control of tumor growth without encountering systemic toxicity. The observation that TCR engineering combined with Tet-regulated IL-12 expression can achieve tumor immunity without the side effects that are usually associated with the in vivo use of IL-12 warrants translation of this concept into the clinic.
ABSTRACT
Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1hi liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.
Subject(s)
Carcinoma, Hepatocellular/therapy , Hepatitis B, Chronic/therapy , Liver Neoplasms/therapy , Programmed Cell Death 1 Receptor/immunology , Receptors, Antigen, T-Cell/therapeutic use , Antigens, Neoplasm/immunology , Antigens, Neoplasm/therapeutic use , Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Genetic Vectors/genetics , Hepatitis B virus/immunology , Hepatitis B virus/pathogenicity , Hepatitis B, Chronic/immunology , Hepatitis B, Chronic/pathology , Hepatitis B, Chronic/virology , Humans , Immunotherapy, Adoptive/methods , Lentivirus/genetics , Liver/immunology , Liver/metabolism , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Liver Neoplasms/virology , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/therapeutic use , Receptors, Antigen, T-Cell/immunology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Ongoing clinical trials explore T cell receptor (TCR) gene therapy as a treatment option for cancer, but responses in solid tumors are hampered by the immunosuppressive microenvironment. The production of TCR gene-engineered T cells requires full T cell activation in vitro, and it is currently unknown whether in vivo interactions with conventional dendritic cells (cDCs) regulate the accumulation and function of engineered T cells in tumors. Using the B16 melanoma model and the inducible depletion of CD11c+ cells in CD11c.diphtheria toxin receptor (DTR) mice, we analyzed the interaction between tumor-resident cDCs and engineered T cells expressing the melanoma-specific TRP-2 TCR. We found that depletion of CD11c+ cells triggered the recruitment of cross-presenting cDC1 into the tumor and enhanced the accumulation of TCR-engineered T cells. We show that the recruited tumor cDCs present melanoma tumor antigen, leading to enhanced activation of TCR-engineered T cells. In addition, detailed analysis of the tumor myeloid compartment revealed that the depletion of a population of DT-sensitive macrophages can contribute to the accumulation of tumor-infiltrating T cells. Together, these data suggest that the relative frequency of tumor-resident cDCs and macrophages may impact the therapeutic efficacy of TCR gene therapy in solid tumors.
Subject(s)
Dendritic Cells/metabolism , Macrophages/metabolism , Receptors, Antigen, T-Cell/metabolism , Animals , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , CD11c Antigen/immunology , CD11c Antigen/metabolism , Heparin-binding EGF-like Growth Factor/immunology , Heparin-binding EGF-like Growth Factor/metabolism , Humans , Immunotherapy, Adoptive/methods , Melanoma, Experimental/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Antigen, T-Cell/immunologyABSTRACT
The CTLA-4 checkpoint regulates the activation of T cells. Individuals with heterozygous mutations in CTLA-4 have a complex phenotype typically characterized by antibody deficiency alongside variable autoimmunity. Despite severe disease in some individuals, others remain largely unaffected with reasons for this variation unknown. We studied a large family carrying a single point mutation in CTLA-4 leading to an amino acid change R75W and compared both unaffected with affected individuals. We measured a variety of features pertaining to T cell and CTLA-4 biology and observed that at the cellular level there was complete penetrance of CTLA-4 mutations. Accordingly, unaffected individuals were indistinguishable from those with disease in terms of level of CTLA-4 expression, percentage of Treg, upregulation of CTLA-4 upon stimulation and proliferation of CD4 T cells. We conclude that the wide variation in disease phenotype is influenced by immune variation outside of CTLA-4 biology.