T cells specific for α-myosin drive immunotherapy-related myocarditis.

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

Therapeutic options for CTLA-4 insufficiency.

BACKGROUND: Heterozygous germline mutations in cytotoxic T lymphocyte-associated antigen-4 (CTLA4) impair the immunomodulatory function of regulatory T cells. Affected individuals are prone to life-threatening autoimmune and lymphoproliferative complications. A number of therapeutic options are currently being used with variable effectiveness. OBJECTIVE: Our aim was to characterize the responsiveness of patients with CTLA-4 insufficiency to specific therapies and provide recommendations for the diagnostic workup and therapy at an organ-specific level. METHODS: Clinical features, laboratory findings, and response to treatment were reviewed retrospectively in an international cohort of 173 carriers of CTLA4 mutation. Patients were followed between 2014 and 2020 for a total of 2624 months from diagnosis. Clinical manifestations were grouped on the basis of organ-specific involvement. Medication use and response were recorded and evaluated. RESULTS: Among the 173 CTLA4 mutation carriers, 123 (71%) had been treated for immune complications. Abatacept, rituximab, sirolimus, and corticosteroids ameliorated disease severity, especially in cases of cytopenias and lymphocytic organ infiltration of the gut, lungs, and central nervous system. Immunoglobulin replacement was effective in prevention of infection. Only 4 of 16 patients (25%) with cytopenia who underwent splenectomy had a sustained clinical response. Cure was achieved with stem cell transplantation in 13 of 18 patients (72%). As a result of the aforementioned methods, organ-specific treatment pathways were developed. CONCLUSION: Systemic immunosuppressants and abatacept may provide partial control but require ongoing administration. Allogeneic hematopoietic stem cell transplantation offers a possible cure for patients with CTLA-4 insufficiency.

Abatacept for treatment-refractory pediatric CTLA4-haploinsufficiency.

CTLA4-haploinsufficiency is a complex disease of immune dysregulation presenting with a broad spectrum of clinical manifestations. CTLA4-Fc fusion proteins such as abatacept have been described to alleviate immune dysregulation in several adult cases of CTLA4-haploinsufficiency. However, until now only few cases of pediatric CTLA4-haploinsufficiency treated with abatacept have been described. Here we present two pediatric cases of severe CTLA4-haploinsufficiency refractory to conventional immunosuppressive therapies that responded rapidly to treatment with abatacept. No side effects were observed during a follow-up period of 7-15 months. While one patient has successfully undergone HSCT the second patient continues to receive abatacept. Our cases demonstrate safe medium-term use of abatacept in the pediatric population.

Gastric cancer, inflammatory bowel disease and polyautoimmunity in a 17-year-old boy: CTLA-4 deficiency successfully treated with Abatacept.

Gut involvement is frequent in immunologic disorders, especially with inflammatory manifestations but also with cancer. In the last years, advances in functional and genetic testing have improved the diagnostic and therapeutic approach to immune dysregulation syndromes. CTLA-4 deficiency is a rare disease with variable phenotype, ranging from absence of symptoms to severe multisystem manifestations and complications. We describe a rare case of CTLA-4 deficiency in a boy with gastric cancer, very early onset inflammatory bowel disease and polyautoimmunity, the second-ever reported in the literature with the same characteristics. A 17-year-old boy was referred to Bambino Gesù Children's Hospital of Rome, a tertiary care center, for a gastric mass and a long-term history of very early onset inflammatory bowel disease, diabetes mellitus type 1, polyarthritis and psoriasis. Histology of gastric biopsies revealed the presence of neoplastic signet ring cells. Imaging staging showed localized cancer; therefore, the patient underwent subtotal gastrectomy with termino-lateral gastro-jejunal anastomosis. Immunological work up and genetic testing by next-generation sequencing panels for primary immunodeficiencies led to the diagnosis of CTLA-4 deficiency. Good disease control was obtained with the administration of Abatacept. The patient experienced an asymptomatic SARS-CoV-2 infection without any concern. Eighteen months after treatment initiation, the patient is alive and well. Immunologic and genetic testing, such as next-generation sequencing, should always be part of the diagnostic approach to patients with complex immune dysregulation syndrome, severe clinical course, poor response to treatments or cancer. The early recognition of the monogenic disease is the key for disease management and targeted therapy.

Underlying CTLA4 Deficiency in a Patient With Juvenile Idiopathic Arthritis and Autoimmune Lymphoproliferative Syndrome Features Successfully Treated With Abatacept-A Case Report.

BACKGROUND: Functional variants of the cytotoxic T-lymphocyte antigen-4 (CTLA4) could contribute to the pathogenesis of disorders characterized by abnormal T-cell responses. CASE PRESENTATION: We report a case of a 13-year-old girl who first presented with polyarticular juvenile idiopathic arthritis poorly responsive to treatment. During the following years the patient developed cytopenias, chronic lymphoproliferation, high values of T-cell receptor αß+ CD4- CD8- double-negative T cells and defective Fas-mediated T cells apoptosis. Autoimmune lymphoproliferative syndrome was diagnosed and therapy with mycophenolate mofetil was started, with good hematological control. Due to the persistence of active polyarthritis, mycophenolate mofetil was replaced with sirolimus. In the following months the patient developed hypogammaglobulinemia and started having severe diarrhea. Histologically, duodenitis and chronic gastritis were present. Using the next generation sequencing-based gene panel screening, a CTLA4 mutation was detected (p.Cys58Serfs*13). At the age of 21 the patient developed acute autoimmune hemolytic anemia; steroid treatment in combination with abatacept were started with clinical remission of all symptoms, even arthritis. CONCLUSIONS: Targeted immunologic screening and appropriate genetic tests could help in the diagnosis of a specific genetically mediated immune dysregulation syndrome, allowing to select those patients who can take advantage of target therapy, as in the case of abatacept in CTLA4 deficiency.

Clinical Courses of IKAROS and CTLA4 Deficiencies: A Systematic Literature Review and Retrospective Longitudinal Study.

IKAROS and CTLA4 deficiencies are inborn errors of immunity and show similar clinical phenotypes, including hypogammaglobulinemia and autoimmune diseases (ADs). However, the differences in clinical features and pathogenesis of these are not fully understood. Therefore, we performed systematic literature reviews for IKAROS and CTLA4 deficiencies. The reviews suggested that patients with IKAROS deficiency develop AD earlier than hypogammaglobulinemia. However, no study assessed the detailed changes in clinical manifestations over time; this was likely due to the cross-sectional nature of the studies. Therefore, we conducted a retrospective longitudinal study on IKAROS and CTLA4 deficiencies in our cohort to evaluate the clinical course over time. In patients with IKAROS deficiency, AD and hypogammaglobulinemia often develop in that order, and AD often resolves before the onset of hypogammaglobulinemia; these observations were not found in patients with CTLA4 deficiency. Understanding this difference in the clinical course helps in the clinical management of both. Furthermore, our results suggest B- and T-cell-mediated ADs in patients with IKAROS and CTLA4 deficiencies, respectively.

Haploinsufficiency of immune checkpoint receptor CTLA4 induces a distinct neuroinflammatory disorder.

BACKGROUNDCytotoxic T lymphocyte antigen 4 (CTLA4) is essential for immune homeostasis. Genetic mutations causing haploinsufficiency (CTLA4h) lead to a phenotypically heterogenous, immune-mediated disease that can include neuroinflammation. The neurological manifestations of CTLA4h are poorly characterized.METHODSWe performed an observational natural history study of 50 patients with CTLA4h who were followed at the NIH. We analyzed clinical, radiological, immunological, and histopathological data.RESULTSEvidence for neuroinflammation was observed in 32% (n = 16 of 50) of patients in this cohort by magnetic resonance imaging (MRI) and/or by cerebrospinal fluid analysis. Clinical symptoms were commonly absent or mild in severity, with headaches as the leading complaint (n = 13 of 16). The most striking findings were relapsing, large, contrast-enhancing focal lesions in the brain and spinal cord observed on MRI. We detected inflammation in the cerebrospinal fluid and leptomeninges before the parenchyma. Brain biopsies of inflammatory lesions from 10 patients showed perivascular and intraparenchymal mixed cellular infiltrates with little accompanying demyelination or neuronal injury.CONCLUSIONSNeuroinflammation due to CTLA4h is mediated primarily by an infiltrative process with a distinct and striking dissociation between clinical symptoms and radiological findings in the majority of patients.FUNDINGNIAID, NIH, Division of Intramural Research, NINDS, NIH, Division of Intramural Research, and the National Multiple Sclerosis Society-American Brain Foundation.TRIAL REGISTRATIONClinicalTrials.gov NCT00001355.

Unusual Late-onset Enteropathy in a Patient With Lipopolysaccharide-responsive Beige-like Anchor Protein Deficiency.

In recent years, monogenic causes of immune dysregulation syndromes, with variable phenotypes, have been documented. Mutations in the lipopolysaccharide-responsive beige-like anchor (LRBA) protein are associated with common variable immunodeficiency, autoimmunity, chronic enteropathy, and immune dysregulation disorders. The LRBA protein prevents degradation of cytotoxic T-lymphocyte antigen 4 (CTLA4) protein, thus inhibiting immune responses. Both LRBA and CTLA4 deficiencies usually present with immune dysregulation, mostly characterized by autoimmunity and lymphoproliferation. In this report, we describe a patient with an atypical clinical onset of LRBA deficiency and the patient's response to abatacept, a fusion protein-drug that mimics the action of CTLA4.

An 18-year-old woman with pulmonary nodules found to have cytotoxic T-lymphocyte-associated antigen 4 deficiency.

An 18-year-old woman was admitted with abdominal pain and hematochezia. She was previously healthy until 15 years of age and was subsequently diagnosed with hypogammaglobulinemia, protein-losing enteropathy, a benign temporal lobe brain lesion/orbital fibroadenoma, autoimmune hepatitis, iron deficiency anaemia and hypothyroidism. She developed respiratory distress and hypoxemia. She was found to have nodules on chest CT scan. She was diagnosed with cytotoxic T-lymphocyte-associated antigen 4 deficiency via genetic testing.

Negative Co-stimulation Constrains T Cell Differentiation by Imposing Boundaries on Possible Cell States.

Co-stimulation regulates T cell activation, but it remains unclear whether co-stimulatory pathways also control T cell differentiation. We used mass cytometry to profile T cells generated in the genetic absence of the negative co-stimulatory molecules CTLA-4 and PD-1. Our data indicate that negative co-stimulation constrains the possible cell states that peripheral T cells can acquire. CTLA-4 imposes major boundaries on CD4+ T cell phenotypes, whereas PD-1 subtly limits CD8+ T cell phenotypes. By computationally reconstructing T cell differentiation paths, we identified protein expression changes that underlied the abnormal phenotypic expansion and pinpointed when lineage choice events occurred during differentiation. Similar alterations in T cell phenotypes were observed after anti-CTLA-4 and anti-PD-1 antibody blockade. These findings implicate negative co-stimulation as a key regulator and determinant of T cell differentiation and suggest that checkpoint blockade might work in part by altering the limits of T cell phenotypes.

[Checkpoint inhibitors neurological side effects]. / Complicaciones neurologicas de los inhibidores de punto de control inmunologico.

INTRODUCTION: Checkpoint inhibitors have dramatically transformed cancer treatment. However, due to the increasing number of tumors in which they are used, there is a high number of reported adverse effects. Among them, we highlight neurological side effects. In the approbatory clinical trials, they were thought to be sparse, but they may have been underestimated. AIM: To review the physiopathology and the incidence of neurological side effects due to the use of checkpoint inhibitors, as well as the clinical practice guidelines published in the last years. DEVELOPMENT: To review the published case reports of neurological side effects since the approval of checkpoint inhibitors, and our own experience. Moreover, we summarize the main clinical practice guidelines. CONCLUSIONS: Checkpoint inhibitors neurological side effects are frequent. A wide variety of central or peripheral nervous system symptoms may develop. In the setting of brain tumors, inflammation due to immune system activation might lead to pseudoprogression. Further studies are needed to better describe these neurological side effects, and to implement clinical guidelines.

TITLE: Complicaciones neurologicas de los inhibidores de punto de control inmunologico.Introduccion. Los inhibidores de punto de control inmunologico han supuesto un nuevo paradigma en el tratamiento de diferentes tipos de neoplasias. Sin embargo, con el uso creciente de estos farmacos, se estan observando diferentes efectos adversos. Entre ellos destacan los neurologicos, puesto que su frecuencia parece haberse infraestimado en los ensayos aprobatorios. Objetivo. Revisar la fisiopatologia y la incidencia de los efectos adversos neurologicos por inhibidores de punto de control neurologicos, asi como el abordaje basandose en diferentes guias clinicas. Desarrollo. Se revisan los casos que se han publicado desde la aprobacion de los farmacos y añadimos la experiencia de nuestro centro. A su vez, se hace un resumen de las diferentes guias publicadas de forma reciente. Conclusiones. Las complicaciones derivadas del uso de los inhibidores de punto de control inmunologico son frecuentes. Incluyen multiples cuadros de diferente gravedad, y pueden afectar a cualquier parte del sistema nervioso central y periferico. Ademas, en tumores del sistema nervioso, puede observarse un fenomeno de pseudoprogresion derivado de la inflamacion asociada. Queda pendiente realizar nuevos estudios para conocer en detalle estos efectos adversos y desarrollar guias clinicas con las que optimizar el manejo.

The role of FOXP3+ regulatory T cells in human autoimmune and inflammatory diseases.

CD4+ regulatory T cells (Treg ) expressing the forkhead box protein 3 (FOXP3) transcription factor (Tregs ) are instrumental for the prevention of autoimmune diseases. There is increasing evidence that the human T regulatory population is highly heterogeneous in phenotype and function. Numerous studies conducted in human autoimmune diseases have shown that Treg cells are impaired either in their suppressive function, in number, or both. However, the contribution of the FOXP3+ Treg subpopulations to the development of autoimmunity has not been delineated in detail. Rare genetic disorders that involve deficits in Treg function can be studied to develop a global idea of the impact of partial or complete deficiency in a specific molecular mechanism involved in Treg function. In patients with reduced Treg numbers (but no functional deficiency), the expansion of autologous Treg cells could be a suitable therapeutic approach: either infusion of in-vitro autologous expanded cells, infusion of interleukin (IL)-2/anti-IL-2 complex, or both. Treg biology-based therapies may not be suitable in patients with deficits of Treg function, unless their deficit can be corrected in vivo/in vitro. Finally, it is critical to consider the appropriate stage of autoimmune diseases at which administration of Treg cellular therapy can be most effective. We discuss conflicting data regarding whether Treg cells are more effectual at preventing the initiation of autoimmunity, ameliorating disease progression or curing autoimmunity itself.

CTLA-4 regulates T follicular regulatory cell differentiation and participates in intestinal damage caused by spontaneous autoimmunity.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is a co-inhibitory molecule expressed by T cells and is required for immune regulation and inflammation prevention. In clinical patients, the CTLA-4 mutation causes spontaneous immune-related early-onset Crohn's disease; however, its potential mechanism is still unknown. In the current study, we found that defects in CTLA-4 in CD4 cells lead to limited differentiation of T follicular regulatory (Tfr) cells and relatively increased T follicular helper (Tfh) cells and spontaneous B cell germinal centres (GCs) responses that trigger the accumulation of autoantibodies in intestinal epithelial cells. In addition, the deficiency of Tfr cells caused by defects in CTLA-4 causes these cells to lose their function of inhibiting the non-specific immune response produced during the specific humoural immune response induced by MCMV (mouse cytomegalovirus), resulting in acute intestinal injury and death in mice. The lack of Tfr cells may be responsible for the immunosuppressive disorder of inflammatory bowel disease caused by CTLA-4 deficiency. In conclusion, we verified that CTLA-4 may be required for Tfr cell differentiation and production. Tfr cells inhibit B cell responses and prevent humoural autoimmune-mediated intestinal damage by regulating Tfh-dependent GC responses.

Study of an extended family with CTLA-4 deficiency suggests a CD28/CTLA-4 independent mechanism responsible for differences in disease manifestations and severity.

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.

Initiation of inflammatory tumorigenesis by CTLA4 insufficiency due to type 2 cytokines.

Genetically predisposed CTLA4 insufficiency in humans is associated with gastric cancer development, which is paradoxical to the prototypical role of CTLA4 in suppressing antitumor immunity. CTLA4 is a critical immune checkpoint against autoimmune disorders. Autoimmunity has been implicated in protumor or antitumor activities. Here, we show that CTLA4 insufficiency initiates de novo tumorigenesis in the mouse stomach through inflammation triggered by host-intrinsic immune dysregulation rather than microbiota, with age-associated progression to malignancy accompanied by epigenetic dysregulation. The inflammatory tumorigenesis required CD4 T cells, but not the TH1 or TH17 subsets. Deficiencies in IL-4 and IL-13 or IL-4 receptor α broke the link between inflammation and initiation of tumorigenesis. This study establishes the causality of CTLA4 insufficiency in gastric cancer and uncovers a role of type 2 inflammation in initiating gastric epithelial transformation. These findings suggest possible improvement of immune therapies by blocking tumorigenic type 2 inflammation while preserving antitumor type 1 immunity.

CTLA-4-mediated posttranslational modifications direct cytotoxic T-lymphocyte differentiation.

The blockade of inhibitory receptors such as CTLA-4 (CD152) is being used as immune-checkpoint therapy, offering a powerful strategy to restore effective immune responses against tumors. To determine signal components that are induced under the control of CTLA-4 we analyzed activated murine CD8+ T cells by quantitative proteomics. Accurate mass spectrometry revealed that CTLA-4 engagement led to central changes in the phosphorylation of proteins involved in T-cell differentiation. Beside other targets, we discovered a CTLA-4-mediated induction of the translational inhibitor programmed cell death-4 (PDCD4) as a result of FoxO1 nuclear re-localization. PDCD4 further bound a distinct set of mRNAs including Glutaminase, which points out a critical role for CTLA-4 in CD8+ T-cell metabolism. Consequently, PDCD4-deficient cytotoxic T-lymphocytes (CTLs) expressed increased amounts of otherwise repressed effector molecules and ultimately led to superior control of tumor growth in vivo. These findings reveal a novel CTLA-4-mediated pathway to attenuate CTLs and indicate the importance of post-transcriptional mechanisms in the regulation of anti-tumor immune responses.

EFIS Lecture: Understanding the CTLA-4 checkpoint in the maintenance of immune homeostasis.

The past 20 years have heralded fascinating developments in the field of CTLA-4 biology. The CTLA-4 protein is a critical negative regulator of T cell immunity and its absence provokes severe lymphoproliferative disease. In a surprising twist, the generation of mixed bone marrow chimeric mice revealed that CTLA-4 predominantly functions in a cell-extrinsic manner, suggesting that CTLA-4 expressed on one cell can modify the behaviour of another cell. This was followed by the demonstration that CTLA-4 is highly expressed in regulatory T cells and can contribute to their suppressive activity. In line with a cell-extrinsic function, increasing evidence indicates that CTLA-4-positive cells can modify the phenotype of antigen presenting cells (APC), thereby regulating the priming of naive T cells. Notably, CTLA-4 is able to downregulate expression of costimulatory ligands on APC via a process of trans-endocytosis. The identification of patients with mutations in the ctla4 gene has provided an opportunity to study the contribution of CTLA-4 to Treg function and immune regulation in the human immune system. Finally, it has become apparent that CTLA-4 also plays a role in controlling humoral immunity, via the regulation of CD28-driven follicular helper T cell differentiation. At the recent German Society for Immunology congress, I discussed some of the contributions of my own lab to the unfolding of the CTLA-4 story, in the context of the work of others in the field. Despite the enormous clinical potential associated with modulation of the CTLA-4 pathway, including the use of soluble CTLA-4 molecules in autoimmune settings and blocking antibodies in cancer, it is clear there is still much to learn about this important pathway.

Induction of autoimmune disease by deletion of CTLA-4 in mice in adulthood.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is essential for immunological (self-) tolerance, but due to the early fatality of CTLA-4 KO mice, its specific function in central and peripheral tolerance and in different systemic diseases remains to be determined. Here, we further examined the role of CTLA-4 by abrogating CTLA-4 expression in adult mice and compared the resulting autoimmunity that follows with that produced by congenital CTLA-4 deficiency. We found that conditional deletion of CTLA-4 in adult mice resulted in spontaneous lymphoproliferation, hypergammaglobulinemia, and histologically evident pneumonitis, gastritis, insulitis, and sialadenitis, accompanied by organ-specific autoantibodies. However, in contrast to congenital deficiency, this was not fatal. CTLA-4 deletion induced preferential expansion of CD4(+)Foxp3(+) Treg cells. However, T cells from CTLA-4-deficient inducible KO mice were able to adoptively transfer the diseases into T cell-deficient mice. Notably, cell transfer of thymocytes de novo produced myocarditis, otherwise not observed in donor mice depleted in adulthood. Moreover, CTLA-4 deletion in adult mice had opposing impacts on induced autoimmune models. Thus, although CTLA-4-deficient mice had more severe collagen-induced arthritis (CIA), they were protected against peptide-induced experimental autoimmune encephalomyelitis (EAE); however, onset of protein-induced EAE was only delayed. Collectively, this indicates that CTLA-4 deficiency affects both central and peripheral tolerance and Treg cell-mediated suppression.