Maintenance Of Self-tolerance Research Articles

Aging and Parkinson’s disease: a complex interplay of vulnerable neurons, the immune system and the blood-brain barrier

Aging is the biggest risk factor for Parkinson’s disease (PD) and a particular vulnerability of dopaminergic neurons in the substantia nigra to aging-associated effects has been firmly established. More recent work has revealed an important role of non-neuronal systems such as the blood-brain barrier (BBB) or the immune system in the pathogenesis of PD. Effects of aging on the immune system include a chronic inflammatory state termed inflammaging and immunosenescence. Both processes are connected to a higher pro-inflammatory potency and negatively affect the maintenance of self-tolerance. The BBB gets increasingly dysfunctional with advancing age and its endothelial cells display a more pro-inflammatory phenotype while the transport of important plasma proteins to the brain is impaired. The immune system and the BBB are heavily interdependent and are both essential for the homeostasis of especially vulnerable dopaminergic neurons. The degeneration of dopaminergic neurons can, in turn, influence the BBB or the immune system, potentially creating a vicious cycle. In this review, we aim to develop a multisystem perspective on aging and PD by incorporating the aging immune system and aging BBB into the pathophysiological processes. Given the current evidence, it seems likely that a combination of multimodal effects of aging on the levels of SN pars compacta (SNc) dopaminergic neurons, the immune system, and the BBB increase the risk of developing PD.

Mechanism study of ubiquitination in T cell development and autoimmune disease.

T cells play critical role in multiple immune processes including antigen response, tumor immunity, inflammation, self-tolerance maintenance and autoimmune diseases et. Fetal liver or bone marrow-derived thymus-seeding progenitors (TSPs) settle in thymus and undergo T cell-lineage commitment, proliferation, T cell receptor (TCR) rearrangement, and thymic selections driven by microenvironment composed of thymic epithelial cells (TEC), dendritic cells (DC), macrophage and B cells, thus generating T cells with diverse TCR repertoire immunocompetent but not self-reactive. Additionally, some self-reactive thymocytes give rise to Treg with the help of TEC and DC, serving for immune tolerance. The sequential proliferation, cell fate decision, and selection during T cell development and self-tolerance establishment are tightly regulated to ensure the proper immune response without autoimmune reaction. There are remarkable progresses in understanding of the regulatory mechanisms regarding ubiquitination in T cell development and the establishment of self-tolerance in the past few years, which holds great potential for further therapeutic interventions in immune-related diseases.

Treatment Options for Hepatocellular Carcinoma Using Immunotherapy: Present and Future.

Hepatocellular carcinoma (HCC) is a common cancer, and the body's immune responses greatly affect its progression and the prognosis of patients. Immunological suppression and the maintenance of self-tolerance in the tumor microenvironment are essential responses, and these form part of the theoretical foundations of immunotherapy. In this review, we first discuss the tumor microenvironment of HCC, describe immunosuppression in HCC, and review the major biomarkers used to track HCC progression and response to treatment. We then examine antibody-based therapies, with a focus on immune checkpoint inhibitors (ICIs), monoclonal antibodies that target key proteins in the immune response (programmed cell death protein 1, anti-cytotoxic T-lymphocyte associated protein 4, and programmed death-ligand 1) which have transformed the treatment of HCC and other cancers. ICIs may be used alone or in conjunction with various targeted therapies for patients with advanced HCC who are receiving first-line treatments or subsequent treatments. We also discuss the use of different cellular immunotherapies, including T cell receptor (TCR) T cell therapy and chimeric antigen receptor (CAR) T cell therapy. We then review the use of HCC vaccines, adjuvant immunotherapy, and oncolytic virotherapy, and describe the goals of future research in the development of treatments for HCC.

The role of immune checkpoints in antitumor response: a potential antitumor immunotherapy.

Immunotherapy aims to stimulate the immune system to inhibit tumor growth or prevent metastases. Tumor cells primarily employ altered expression of human leukocyte antigen (HLA) as a mechanism to avoid immune recognition and antitumor immune response. The antitumor immune response is primarily mediated by CD8+ cytotoxic T cells (CTLs) and natural killer (NK) cells, which plays a key role in the overall anti-tumor immune response. It is crucial to comprehend the molecular events occurring during the activation and subsequent regulation of these cell populations. The interaction between antigenic peptides presented on HLA-I molecules and the T-cell receptor (TCR) constitutes the initial signal required for T cell activation. Once activated, in physiologic circumstances, immune checkpoint expression by T cells suppress T cell effector functions when the antigen is removed, to ensures the maintenance of self-tolerance, immune homeostasis, and prevention of autoimmunity. However, in cancer, the overexpression of these molecules represents a common method through which tumor cells evade immune surveillance. Numerous therapeutic antibodies have been developed to inhibit immune checkpoints, demonstrating antitumor activity with fewer side effects compared to traditional chemotherapy. Nevertheless, it's worth noting that many immune checkpoint expressions occur after T cell activation and consequently, altered HLA expression on tumor cells could diminish the clinical efficacy of these antibodies. This review provides an in-depth exploration of immune checkpoint molecules, their corresponding blocking antibodies, and their clinical applications.

Mouse Tissue-Resident Peritoneal Macrophages in Homeostasis, Repair, Infection, and Tumor Metastasis.

Large peritoneal macrophages (LPMs) are long-lived, tissue-resident macrophages, formed during embryonic life, developmentally and functionally confined to the peritoneal cavity. LPMs provide the first line of defense against life-threatening pathologies of the peritoneal cavity, such as abdominal sepsis, peritoneal metastatic tumor growth, or peritoneal injuries caused by trauma, or abdominal surgery. Apart from their primary phagocytic function, reminiscent of primitive defense mechanisms sustained by coelomocytes in the coelomic cavity of invertebrates, LPMs fulfill an essential homeostatic function by achieving an efficient clearance of apoptotic, that is crucial for the maintenance of self-tolerance. Research performed over the last few years, in mice, has unveiled the mechanisms by which LPMs fulfill a crucial role in repairing peritoneal injuries and controlling microbial and parasitic infections, reflecting that the GATA6-driven LPM transcriptional program can be modulated by extracellular signals associated with pathological conditions. In contrast, recent experimental evidence supports that peritoneal tumors can subvert LPM metabolism and function, leading to the acquisition of a tumor-promoting potential. The remarkable functional plasticity of LPMs can be nevertheless exploited to revert tumor-induced LPM protumor potential, providing the basis for the development of novel immunotherapeutic approaches against peritoneal tumor metastasis based on macrophage reprogramming.

Prolidase Deficiency Causes Spontaneous T Cell Activation and Lupus-like Autoimmunity.

Prolidase deficiency (PD) is a multisystem disorder caused by mutations in the PEPD gene, which encodes a ubiquitously expressed metallopeptidase essential for the hydrolysis of dipeptides containing C-terminal proline or hydroxyproline. PD typically presents in childhood with developmental delay, skin ulcers, recurrent infections, and, in some patients, autoimmune features that can mimic systemic lupus erythematosus. The basis for the autoimmune association is uncertain, but might be due to self-antigen exposure with tissue damage, or indirectly driven by chronic infection and microbial burden. In this study, we address the question of causation and show that Pepd-null mice have increased antinuclear autoantibodies and raised serum IgA, accompanied by kidney immune complex deposition, consistent with a systemic lupus erythematosus-like disease. These features are associated with an accumulation of CD4 and CD8 effector T cells in the spleen and liver. Pepd deficiency leads to spontaneous T cell activation and proliferation into the effector subset, which is cell intrinsic and independent of Ag receptor specificity or antigenic stimulation. However, an increase in KLRG1+ effector CD8 cells is not observed in mixed chimeras, in which the autoimmune phenotype is also absent. Our findings link autoimmune susceptibility in PD to spontaneous T cell dysfunction, likely to be acting in combination with immune activators that lie outside the hemopoietic system but result from the abnormal metabolism or loss of nonenzymatic prolidase function. This knowledge provides insight into the role of prolidase in the maintenance of self-tolerance and highlights the importance of treatment to control T cell activation.

What's new in the pathogeneses and triggering factors of bullous pemphigoid.

Bullous pemphigoid (BP) is a subepidermal blistering disease induced by autoantibodies to type XVII collagen (COL17, also called BP180) and BP230. Previous studies using patients' samples and animal disease models elucidated the complement-dependent and complement-independent pathways of blister formation, the pathogenic roles of immune cells (T and B cells, macrophages, mast cells, neutrophils, eosinophils), and the pathogenicity of IgE autoantibodies in BP. This review introduces the recent progress on the mechanism behind the epitope-spreading phenomenon in BP, which is considered to be important to understand the chronic and intractable disease course of BP, and the pathogenicity of anti-BP230 autoantibodies, mainly focusing on studies that used active disease models. To clarify the pathogenesis of BP, the mechanism behind the breakdown of immune tolerance to BP antigens should be investigated. Recent studies using various experimental models have revealed important roles for regulatory T cells in the maintenance of self-tolerance to COL17 and BP230 as well as in the suppression of inflammation triggered by the binding of antibodies to COL17. Notably, physical stresses such as trauma, thermal burns, bone fractures, irradiation and ultraviolet exposure, some pathologic conditions such as neurological diseases and hematological malignancies, and the use of dipeptidyl peptidase-IV inhibitors and immune checkpoint inhibitors have been reported as triggering factors for BP. These factors and certain underlying conditions such as genetic background, regulatory T-cell dysfunction or aging might synergistically affect some individuals and eventually induce BP. Further studies on the breakdown of self-tolerance and on the identification of key molecules that are relevant to blister formation and inflammation may expand our understanding of BP's etiology and may lead to the development of novel therapeutic approaches.

Analysis of FOXP3 DNA Methylation Patterns to Identify Functional FOXP3+ T-Cell Subpopulations.

Human regulatory CD4+CD25+FOXP3+ T cells (Tregs) are involved in the suppression of immune responses and play important roles in the maintenance of self-tolerance and immune homeostasis. Abnormal Treg function may result in disease states of varying severity. As FOXP3-expressing Treg cells are phenotypically and functionally heterogeneous, the success of Treg therapies depends on the ability to reliably distinguish subpopulations of T cells bearing a Treg-like phenotype. Methylation of cytosines within CpG dinucleotides is an important epigenetic mechanism involved in regulation (and suppression) of gene expression. On the other hand, demethylation of regulatory DNA sequences, such as promoters and enhancers, is essential for initiation of gene transcription. This protocol shows that bisulfite sequencing (BS) distinguishes methylated and unmethylated cytosines within DNA and reveals the methylation status of individual CpGs in cells within each population, identifying functionally different FOXP3+ subpopulations.

The Neonatal Innate Immune Response to Sepsis: Checkpoint Proteins as Novel Mediators of This Response and as Possible Therapeutic/Diagnostic Levers.

Sepsis, a dysfunctional immune response to infection leading to life-threatening organ injury, represents a significant global health issue. Neonatal sepsis is disproportionately prevalent and has a cost burden of 2-3 times that of adult patients. Despite this, no widely accepted definition for neonatal sepsis or recommendations for management exist and those created for pediatric patients are significantly limited in their applicability to this unique population. This is in part due to neonates’ reliance on an innate immune response (which is developmentally more prominent in the neonate than the immature adaptive immune response) carried out by dysfunctional immune cells, including neutrophils, antigen-presenting cells such as macrophages/monocytes, dendritic cells, etc., natural killer cells, and innate lymphoid regulatory cell sub-sets like iNKT cells, γδ T-cells, etc.Immune checkpoint inhibitors are a family of proteins with primarily suppressive/inhibitory effects on immune and tumor cells and allow for the maintenance of self-tolerance. During sepsis, these proteins are often upregulated and are thought to contribute to the long-term immunosuppression seen in adult patients. Several drugs targeting checkpoint inhibitors, including PD-1 and PD-L1, have been developed and approved for the treatment of various cancers, but no such therapeutics have been approved for the management of sepsis. In this review, we will comparatively discuss the role of several checkpoint inhibitor proteins, including PD-1, PD-L1, VISTA, and HVEM, in the immune response to sepsis in both adults and neonates, as well as posit how they may uniquely propagate their actions through the neonatal innate immune response. We will also consider the possibility of leveraging these proteins in the clinical setting as potential therapeutics/diagnostics that might aid in mitigating neonatal septic morbidity/mortality.

SCARF1-Induced Efferocytosis Plays an Immunomodulatory Role in Humans, and Autoantibodies Targeting SCARF1 Are Produced in Patients with Systemic Lupus Erythematosus.

Deficiency in the clearance of cellular debris is a major pathogenic factor in the emergence of autoimmune diseases. We previously demonstrated that mice deficient for scavenger receptor class F member 1 (SCARF1) develop a lupus-like autoimmune disease with symptoms similar to human systemic lupus erythematosus (SLE), including a pronounced accumulation of apoptotic cells (ACs). Therefore, we hypothesized that SCARF1 will be important for clearance of ACs and maintenance of self-tolerance in humans, and that dysregulation of this process could contribute to SLE. In this article, we show that SCARF1 is highly expressed on phagocytic cells, where it functions as an efferocytosis receptor. In healthy individuals, we discovered that engagement of SCARF1 by ACs on BDCA1+ dendritic cells initiates an IL-10 anti-inflammatory response mediated by the phosphorylation of STAT1 and STAT3. Unexpectedly, there was no significant difference in SCARF1 expression in samples of patients with SLE compared with healthy donor samples. However, we detected anti-SCARF1 autoantibodies in 26% of patients with SLE, which was associated with dsDNA Ab positivity. Furthermore, our data show a direct correlation of the levels of anti-SCARF1 in the serum and defects in the removal of ACs. Depletion of Ig restores efferocytosis in SLE serum, suggesting that defects in the removal of ACs are partially mediated by SCARF1 pathogenic autoantibodies. Our data demonstrate that human SCARF1 is an AC receptor in dendritic cells and plays a role in maintaining tolerance and homeostasis.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product

BackgroundA growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype).ResultsThe GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function.ConclusionsThese results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

Prevention of Acute Graft-Versus-Host Disease Using an Engineered Mouse Orthogonal IL-2/IL-2Rβ Regulatory T Cells

Regulatory T cells are a critical population for the maintenance of self-tolerance and the use of these cells for therapeutic purpose in transplantation has emerged based on their capacity to control immune activation, exhibiting positive therapeutic potential to ameliorate acute graft-versus-host disease (GvHD), one of the major complications after allogeneic bone marrow transplantation (allo-BMT). One of the remaining technical challenges in the clinical practice of Treg administration is the number of Treg required to effectively prevent GvHD. In this study, we engineered murine Treg cells to express an altered IL-2 receptor β subunit that selectively interacts with an orthogonal mutant IL-2 (oIL2) without interacting with the natural cytokine or receptor counterparts, thus preventing acute -GvHD.For investigation of the suppressive function of Treg orthogonal IL-2/IL-2 receptor system in a murine major mismatch of acute GvHD model, the BALB/c mice were lethally irradiated (8.8 Gy) and transplanted with 5x10 6 T cell-depleted bone marrow cells (TCD-BM) from C57Bl/6 mice alone or together with fresh 1x10 6 C57Bl/6 FoxP3 DRT-GFP-luc+ Treg cells (fTreg) or with 0.2x10 6 and 1x10 6 oTreg (transduced with the construct for murine oIL-2 receptor β chain) on day 0. On day 2, C57Bl/6 Tcon cells (1x10 6) were injected to induce GvHD. Recipients were treated with PBS or oIL-2 (25K IU/daily) for 14 days and then 3 times a week for another 14 days.Using bioluminescence imaging, we observed that ortho IL2Rβ +Treg not only maintains migration ability to the gut, mLN and LN but also exhibited significant expansion ability treated with oIL2 at day 7 post-transplant. The expansion of oTreg with PBS and fTreg was observed on day 14 after allo-BMT. The administration of oTreg-oIL2 and fTreg (1:1 Tcon:Treg ratio) significantly increased the survival of the mice compared to Tcon group (P<0.0001 and P=0.0001, respectively). Furthermore, the group oTreg-oIL2 (1:1) showed a significant increase in the survival curve compared to the fTreg group (P=0.03). When decreasing the number of oTreg administrated (5:1 Tcon:Treg ratio) a significant increase in the survival (Tcon vs Tcon:oTreg (5:1)+oIL2; P<0.0001) in the oIL2 group. Similar survival curves were observed between this group and fTreg group (1:1).oIL-2 protein injection was able to selectively expand the oTreg in vivo, with a significantly increased percentage of Foxp3 GFP+ in CD4 + T cells (PBS;14.96±2.4%, oIL-2; 27.76±23.6%, P=0.02) in the peripheral blood (PB) and spleen after 7 days post-transplant. We also observed a significant decrease in the percentage of effector T cells in the LN, mLN and spleen of the mice transplanted with oTreg and oIL-2 administration after 7 days post transplantation compared with GvHD group. We further demonstrated that ortho-IL2 administration-maintained graft-versus-leukemia (GvL) responses against lymphoma cells (A20).Engineered orthogonal cytokine receptor improves the suppressing abilities of Treg potential in a murine model of acute GvHD with the ability to maintain GvL, representing a novel approach to the utilization of Treg in Allogeneic Hematopoietic Stem Cell Transplant. DisclosuresGarcia: Synthekine: Membership on an entity's Board of Directors or advisory committees. Blazar: Tmunity Therapeutics: Other: Co-founder; Equilibre Pharmaceuticals Corp: Research Funding; Carisma Therapeutics, Inc: Research Funding; Rheos Medicines: Research Funding; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

TGF-β-induced CD4+ FoxP3+ regulatory T cell-derived extracellular vesicles modulate Notch1 signaling through miR-449a and prevent collagen-induced arthritis in a murine model.

CD4+FOXP3+ Treg cells are central to the maintenance of self-tolerance and can be defective in autoimmunity. In autoimmune rheumatic diseases, dysfunctional self-tolerance, is to a large extent, caused by insufficient Treg-cell activity. Although nTregs have therapeutic effects in vivo, their relative scarcity and slow rate of in vitro expansion hinder the application of nTreg therapy. It was previously reported that EVs contribute significantly to the suppressive function of FOXP3+ Treg cells. Considering that the stability and plasticity of nTregs remain major challenges in vivo, we established EVs derived from in vitro TGF-β-induced Treg cells (iTreg-EVs) and assessed their functions in a murine model of autoimmune arthritis. The results demonstrated that iTreg-EVs preferentially homed to the pathological joint and efficiently prevented the imbalance in Th17/Treg cells in arthritic mice. Furthermore, we found that miR-449a-5p mediated Notch1 expression modulation and that miR-449a-5p knockdown abolished the effects of iTreg-EVs on effector T cells and regulatory T cells in vitro and in vivo. Taken together, our results show that iTreg-EVs control the inflammatory responses of recipient T cells through miR-449a-5p-dependent modulation of Notch1 and ameliorate the development and severity of arthritis, which may provide a potential cell-free strategy based on manipulating iTreg-EVs to prevent autoimmune arthritis.

Coreceptor therapy has distinct short- and long-term tolerogenic effects intrinsic to autoreactive effector T cells.

Immunotherapies are needed in the clinic that effectively suppress β cell autoimmunity and reestablish long-term self-tolerance in type 1 diabetes. We previously demonstrated that nondepleting anti-CD4 (αCD4) and αCD8α antibodies establish rapid and indefinite remission in recent-onset diabetic NOD mice. Diabetes reversal by coreceptor therapy (CoRT) is induced by suppression of pathogenic effector T cells (Teffs) and the selective egress of T cells from the pancreatic lymph nodes and islets that remain free of infiltration in the long term. Here, we defined CoRT-induced events regulating early Teff function and pancreatic residency, and long-term tolerance. TCR-driven gene expression controlling autoreactive Teff expansion and proinflammatory activity was suppressed by CoRT, and islet T cell egress was dependent on sphingosine-1 phosphate. In both murine and human T cells, CoRT upregulated the Foxo1 transcriptional axis, which in turn was required for suppression and efficient pancreatic egress of Teffs. Interestingly, long-term tolerance induced in late-preclinical NOD mice was marked by reseeding of the pancreas by a reduced CD8+ Teff pool exhibiting an exhausted phenotype. Notably, PD-1 blockade, which rescues exhausted Teffs, resulted in diabetes onset in protected animals. These findings demonstrate that CoRT has distinct intrinsic effects on Teffs that impact events early in induction and later in maintenance of self-tolerance.

Immune checkpoints and the multiple faces of B cells in systemic lupus erythematosus.

B-lymphocytes are crucial in the pathogenesis of systemic lupus erythematosus (SLE), including autoantibody production, antigen presentation, co-stimulation, and cytokine secretion. Co-stimulatory and co-inhibitory molecules control interactions between B and T cells during an inflammatory response, which is essential for an appropriate host protection and maintenance of self-tolerance. Here, we review recent findings about checkpoint molecules and SLE B cells including their potential therapeutic implications and experiences from clinical trials. Most prominent checkpoint molecules involved in pathologic B and T cell interaction in SLE are CD40/CD40L and inducible co-stimulator/ICOSL, both also intimately involved in the formation of germinal centers and ectopic lymphoid tissue. Dysregulations of inhibitory checkpoint molecules, like programmed death-1/programmed death-ligand 1 and B- and T-lymphocyte attenuator have been suggested to impair B cell functions in SLE recently. Accumulating evidence indicates that dampening immune responses by either blocking co-activating signals or enhancing co-inhibitory signals in different cell types is a promising approach to treat autoimmune diseases to better control active disease but may also allow resolution of chronic autoimmunity.

Low frequency of CD4+CD25highFOXP3+ T regulatory cells in Egyptian patients with chronic spontaneous urticaria

Chronic urticaria is a prevalent disabling dermatological disease. About 90%, are considered idiopathic and referred to as chronic spontaneous urticaria (CSU), and nearly half of them are likely to have autoimmune mechanisms. Regulatory T cells play a substantial role to prevent autoimmune diseases. Subsets of Tregs expressing the CD4+CD25high and forkhead-box-P3 (FOXP3) transcription factor, crucial for their development and function, are best characterized in maintenance of self-tolerance. The objective of this study was the analysis of peripheral CD4+CD25highFOXP3+(T regs) frequency in chronic spontaneous urticaria; and its possible association with autologous serum skin test (ASST). Fifty chronic spontaneous urticaria patients (25 with positive ASST and 25 with negative ASST) and 20 healthy controls were enrolled in this study. The frequency of CD4+CD25highFOXP3+ (T regs) was analyzed by flow cytometry. A Significant decrease in peripheral blood CD4+CD25highFOXP3+ T regs% was detected in CSU patients in comparison to healthy individuals (median [IQR], 1.47% [0.71-3.12] vs 1.79% [1.15-4.00]; P = 0.05). When ASST positive patients were compared with ASST negative patients, no significant difference was found in percentage of T regs, (P=0.112). In conclusion our data provided further insights into CSU pathogenesis. Reduced frequency of CD4+CD25highFOXP3+(Tregs) in patients with urticaria, support the notion that CSU is an immune mediated disease and may help researchers to develop a novel immunotherapy strategy.

The Tumor Microenvironment Factors That Promote Resistance to Immune Checkpoint Blockade Therapy.

Through genetic and epigenetic alterations, cancer cells present the immune system with a diversity of antigens or neoantigens, which the organism must distinguish from self. The immune system responds to neoantigens by activating naïve T cells, which mount an anticancer cytotoxic response. T cell activation begins when the T cell receptor (TCR) interacts with the antigen, which is displayed by the major histocompatibility complex (MHC) on antigen-presenting cells (APCs). Subsequently, accessory stimulatory or inhibitory molecules transduce a secondary signal in concert with the TCR/antigen mediated stimulus. These molecules serve to modulate the activation signal’s strength at the immune synapse. Therefore, the activation signal’s optimum amplitude is maintained by a balance between the costimulatory and inhibitory signals. This system comprises the so-called immune checkpoints such as the programmed cell death (PD-1) and Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and is crucial for the maintenance of self-tolerance. Cancers often evade the intrinsic anti-tumor activity present in normal physiology primarily by the downregulation of T cell activation. The blockade of the immune checkpoint inhibitors using specific monoclonal antibodies has emerged as a potentially powerful anticancer therapy strategy. Several drugs have been approved mainly for solid tumors. However, it has emerged that there are innate and acquired mechanisms by which resistance is developed against these therapies. Some of these are tumor-intrinsic mechanisms, while others are tumor-extrinsic whereby the microenvironment may have innate or acquired resistance to checkpoint inhibitors. This review article will examine mechanisms by which resistance is mounted against immune checkpoint inhibitors focussing on anti-CTL4-A and anti-PD-1/PD-Ll since drugs targeting these checkpoints are the most developed.

Bim Regulates the Survival and Suppressive Capability of CD8+ Foxp3+ Regulatory T Cells during Graft Versus Host Disease

Graft versus host disease (GVHD) is a proinflammatory syndrome that is the major complication of allogeneic hematopoietic stem cell transplantation and can lead to significant pathological damage. The failure to reconstitute an effective regulatory T cell compartment is a defining characteristic of this disease and has been the genesis for strategies designed to augment reconstitution of regulatory T cells (Tregs) by either in vivo expansion or adoptive transfer of this population. While CD4+ Foxp3+ T cells have been the most carefully examined Treg population, CD8+ Foxp3+ Tregs also emerge early during GVHD, have suppressive function, and can attenuate disease severity. The precise role of these cells and the extent to which CD8+ Tregs possess functional and ontological similarities to CD4+ Tregs, however, have not been well delineated. Using established murine models of GVHD, we observed that adoptively transferred alloantigen-specific CD8+ Tregs had reduced in vivo survival in all target organs and were significantly less potent than CD4+ Tregs for the prevention of lethal GVHD. To define the mechanistic basis for their reduced efficacy, we first examined the T cell receptor (TCR) repertoire of in vivo-derived CD4+ and CD8+ Tregs to determine whether a restricted repertoire was a potential explanation for the diminished ability of CD8+ Tregs to prevent GVHD. We employed GVHD models in which both polyclonal CD8+ [B6→Balb/c] and alloantigen-specific [B6→Balb.B] CD8+ H60+ Treg populations could be assessed and used the Shannon entropy index to define TCR repertoire diversity which is critical for the maintenance of self-tolerance by Tregs. Amplicon sequencing of TCR-β rearrangements to analyze the TCR repertoires of in vivo-induced CD8+ and CD4+ Tregs, along with their conventional CD8+ and CD4+ T cell counterparts, revealed that polyclonal CD8+ and alloantigen-specific CD8+ H60+ Tregs had repertoire diversity that was similar to that of conventional CD8+ T cells, indicating that a more restricted repertoire was not the proximate cause of decreased suppressive capability. Subsequent transcriptional profiling and principal component analysis revealed that CD8+ Tregs arising during GVHD possessed a canonical Treg transcriptional signature that was similar to that observed in CD4+ Tregs, yet distinct from conventional CD8+ T cells. Gene set enrichment and Ingenuity pathway analyses, however, both demonstrated that CD8+ Tregs had differential gene expression in pathways involved in cell death and survival when compared to CD4+ Tregs. This was further confirmed by gene expression analysis and protein expression studies which revealed that CD8+ Tregs had increased expression of the pro-apoptotic proteins Bim, cleaved caspase 3, and cleaved caspase 7, as well as a reduction in the anti-apoptotic protein, Mcl-1. To determine whether Bim was functionally important for CD8+ Treg survival, we comparatively examined in vivo-derived CD4+ and CD8+ Treg induction using wild type Foxp3EGFP and Bim-/-Foxp3EGFP animals as donors in transplantation studies. The absence of Bim in the CD8+ Treg compartment resulted in a significant increase in absolute CD8+ Treg numbers in the liver, lung, and colon when examined on days 7, 14, and 21 post transplantation. Given that the absence of Bim augmented in vivo-generated CD8+ Treg survival, we then examined whether Bim also regulated the survival of adoptively transferred, alloantigen-specific CD8+ Tregs which we had observed were inferior to CD4+ Tregs for the suppression of GVHD. We noted an increased percentage and absolute number of adoptively transferred CD8+ Foxp3+ Bim-/- Tregs relative to wild type CD8+ Tregs in all tissue sites, indicating that the absence of Bim resulted in enhanced stabilization of Foxp3 expression and augmented in vivo survival. Finally, animals that were transplanted with in vitro-derived, alloantigen-specific CD8+ Tregs from Bim-/- Foxp3EGFP mice had significantly prolonged survival relative to mice reconstituted with wild type CD8+ Foxp3EGFP Tregs, demonstrating that the prolonged in vivo survival conferred by the absence of Bim resulted in an increased ability to prevent GVHD. Collectively, these studies demonstrate that Bim plays a critical role in the regulation of CD8+ Treg survival which may have clinical implications for the use of regulatory T cell therapy in the treatment and prevention of GVHD. DisclosuresVincent:Merck: Research Funding; Pharmacyclics: Research Funding; Nanostring: Membership on an entity's Board of Directors or advisory committees. Serody:Merck: Research Funding.

Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor.

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

CMTM6-Deficient Monocytes in ANCA-Associated Vasculitis Fail to Present the Immune Checkpoint PD-L1.

ObjectivesANCA-associated vasculitides (AAV) affect small- and medium-sized blood vessels. In active disease, vessel wall infiltrates are mainly composed of monocytes and macrophages. Immune checkpoint molecules are crucial for the maintenance of self-tolerance and the prevention of autoimmune diseases. After checkpoint inhibitor therapy, the development of autoimmune vasculitis has been observed. However, defects of immune checkpoint molecules in AAV patients have not been identified yet.MethodsMonocytes and monocyte-derived macrophages from AAV patients and healthy age-matched controls were tested for surface expression of immunoinhibitory checkpoint programmed cell death ligand-1 (PD-L1). Using in vitro co-culture approaches, the effect of monocyte PD-L1 expression on CD4+ T cell activation and proliferation was tested.ResultsMonocytes from AAV patients displayed lower PD-L1 expression and a defective PD-L1 presentation upon activation, an effect that was correlated with disease activity. Lower PD-L1 expression was due to increased lysosomal degradation of PD-L1 in AAV monocytes. We identified a reduced expression of CMTM6, a protein protecting PD-L1 from lysosomal breakdown, as the underlying molecular defect. PD-L1low AAV monocytes showed increased stimulatory capacity and induced T cell activation and proliferation. Inhibiting lysosomal function corrected this phenotype by increasing PD-L1, thus normalizing the pro-stimulatory behavior of AAV monocytes.ConclusionsThis study identifies a defect of the immunoinhibitory checkpoint PD-L1 in monocytes from patients with AAV. Low expression of CMTM6 results in enhanced lysosomal degradation of PD-L1, thus providing insufficient negative signaling to T cells. Correcting this defect by targeting lysosomal function may represent a novel strategy to treat AAV.