Bridging Insights From Lymph Node and Synovium Studies in Early Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknown etiology characterized by inflammation of the peripheral synovial joints leading to pannus formation and bone destruction. Rheumatoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) are present years before clinical manifestations and are indicative of a break in tolerance that precedes chronic inflammation. The majority of studies investigating disease pathogenesis focus on the synovial joint as target site of inflammation while few studies explore the initial break in peripheral tolerance which occurs within secondary lymphoid organs such as lymph nodes. If explored during the earliest phases of RA, lymph node research may provide innovative drug targets for disease modulation or prevention. RA research largely centers on the role and origin of lymphocytes, such as pro-inflammatory T cells and macrophages that infiltrate the joint, as well as growing efforts to determine the role of stromal cells within the synovium. It is therefore important to explore these cell types also within the lymph node as a number of mouse studies suggest a prominent immunomodulatory role for lymph node stromal cells. Synovium and proximal peripheral lymph nodes should be investigated in conjunction with one another to gain understanding of the immunological processes driving RA progression from systemic autoimmunity toward synovial inflammation. This perspective seeks to provide an overview of current literature concerning the immunological changes present within lymph nodes and synovium during early RA. It will also propose areas that warrant further exploration with the aim to uncover novel targets to prevent disease progression.

Bridging the Gap: Connecting the Mechanisms of Immune-Related Adverse Events and Autoimmunity Through PD-1

The emergence of anti–cytotoxic T-lymphocyte antigen 4 (anti-CTLA-4), anti–programmed cell death 1 ligand (anti–PD-1), and anti–PD-L1 antibodies as immune checkpoint inhibitors (ICIs) revolutionized the treatment of numerous types of tumors. These antibodies, both alone and in combination, provide great clinical efficacy as evidenced by tumor regression and increased overall patients’ survival. However, with this success comes multiple challenges. First, while patients who respond to ICIs have outstanding outcomes, there remains a large proportion of patients who do not respond at all. This all-or-none response has led to looking downstream of programmed cell death 1 (PD-1) for additional therapeutic targets and for new combination therapies. Second, a majority of patients who receive ICIs go on to develop immune-related adverse events (irAEs) characterized by end-organ inflammation with T-cell infiltrates. The hallmarks of these clinically observed irAEs share many similarities with primary autoimmune diseases. The contribution of PD-1 to peripheral tolerance is a major mechanism for protection against expansion of self-reactive T-cell clones and autoimmune disease. In this review, we aim to bridge the gaps between our cellular and molecular knowledge of PD-1 signaling in T cells, ICI-induced irAEs, and autoimmune diseases. We will highlight shared mechanisms and the potential for new therapeutic strategies.

Pathogenetic value of cell infiltrate in immunoinflammatory rheumatic diseases

Cell infiltrate is a morphological substrate of immunoinflammatory rheumatic diseases. The systemic wide progressive disorganization of loose fibrous connective tissue is accompanied by the loss of tolerance with its own autoantigenes, activation of macrophagal-monocyte cells and autoreactive clones of T and B lymphocytes. Hyperproduction of pro-inflammatory chemokines and cytokines, local adhesive ligandreceptor interactions, endothelial reaction and angiogenesis contribute to the formation of cell infiltrate, ectopic lymphoid structures and GZT-granulomas in situ. The autoimmune response is the result of successive systemic and local molecular cellular events in which the mechanisms of congenital and adaptive immunity are involved. When interpreting immunopathogenesis of rheumatic diseases, all models and schemes adopted in the field of fundamental immunology are used. This is a model of MHC-restrictions, a model of molecular mimicry, or cross of the antigen presentation, a model of disrupting central or peripheral tolerance to auto-antigens, a model of candidate “triggers” of autoimmune and autoinflammatory processes, a model of associations of alleles MHC I and II classes with specific, nosologically unique, rheumatic diseases.

B Cells in Primary Membranous Nephropathy: Escape from Immune Tolerance and Implications for Patient Management

Membranous nephropathy (MN) is an important cause of nephrotic syndrome and chronic kidney disease (CKD) in adults. The pathogenic significance of B cells in MN is increasingly recognized, especially following the discovery of various autoantibodies that target specific podocytic antigens and the promising treatment responses seen with B cell depleting therapies. The presence of autoreactive B cells and autoantibodies that bind to antigens on podocyte surfaces are characteristic features of MN, and are the result of breaches in central and peripheral tolerance of B lymphocytes. These perturbations in B cell tolerance include altered B lymphocyte subsets, dysregulation of genes that govern immunoglobulin production, aberrant somatic hypermutation and co-stimulatory signalling, abnormal expression of B cell-related cytokines, and increased B cell infiltrates and organized tertiary lymphoid structures within the kidneys. An understanding of the role of B cell tolerance and homeostasis may have important implications for patient management in MN, as conventional immunosuppressive treatments and novel B cell-targeted therapies show distinct effects on proliferation, differentiation and reconstitution in different B cell subsets. Circulating B lymphocytes and related cytokines may serve as potential biomarkers for treatment selection, monitoring of therapeutic response and prediction of disease relapse. These recent advances in the understanding of B cell tolerance in MN have provided greater insight into its immunopathogenesis and potential novel strategies for disease monitoring and treatment.

Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus

Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor–transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3−/− mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

255 Investigating VISTA’s role intrinsic to T cells in the tumor microenvironment

BackgroundCancer immunotherapies, specifically checkpoint blockade therapies, have demonstrated clinical importance for long term patient survival. One of the major limitations to checkpoint blockade therapies, is the low response rate: ~30% with anti-CTLA4 and anti-PD1 treatment. This may be due to heterogeneity of the patients‘ immune system and the tumor microenvironment including T cell inhibitions. There is a clear need to study this phenomenon and develop additional therapies for long term survival to include a broad range of patients. V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) is a suppressive protein expressed on many cell types in the tumor microenvironment including cytotoxic T cells. VISTA’s role on T cells has been described as maintaining quiescence and peripheral tolerance in a graft vs host disease model, but is not fully understood in context of the tumor microenvironment.MethodsWe use a series of invivo experiments, including T cell specific VISTA knock outs, to understand the role of VISTA on T cells in the tumor microenvironment.ResultsHere we show a series of in vivo experiments that suggest VISTA has a potent intrinsic role on T cells and therefore anti-tumor immunity. Using a T cell specific VISTA knock out, our results suggest that the absence of VISTA on T cells in combination with anti-CTLA4 and vaccine is a very powerful tumor suppressor compared to vaccine and anti-CTLA4 treatment alone. These results also indicate that the absence of VISTA alters the phenotype of cytotoxic T cells in several ways including the production of inflammatory cytokines.ConclusionsOur preliminary data provides foundation to study VISTA’s role intrinsic to T cells in the tumor microenvironment and how disrupting VISTA’s influence intrinsic to T cells may be advantageous for anti-tumor immunity and long term patient survival.Ethics ApprovalAll in vivo studies were reviewed and approved by Institutional Animal Care and Use Committee (Approval number 2019–2142).

263 ASD141, an innate checkpoint inhibitor, modulates tumor associated myeloid cells through CD11b and enhances current immune checkpoint blockade in preclinical model

BackgroundTumor-associated myeloid cells (TAMCs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). They contribute to immunosuppression and growth of solid tumor. These myeloid cells are highly expressed with CD11b, the alpha-chain of integrin receptor alphaMbeta2 (also known as CD11b/CD18, Mac-1, CR3). It has been suggested that activation of CD11b could facilitate the development of peripheral tolerance by inhibiting T helper 17 differentiation. Antigen-presenting cells (dendritic cells and macrophages) have been shown to enhance T cell proliferation with the treatment of anti-CD11b antibody. Furthermore, CD11b plays a critical role in inflammation by modulating Toll-Like receptor (TLR) responses. High avidity activated form of CD11b leads to a rapid inhibition of TLR signaling by promoting degradation of MyD88 and TRIFs. Therefore, CD11b may serve as an innate checkpoint that function as a negative immune regulator.MethodsIn order to investigate the impact of CD11b in modulating the TME and tumor growth, ASCENDO Biotechnology generated a surrogate chimeric mouse IgG1 antibody, mouse ASD141 (Xi2396), which targets mouse CD11b. These antibodies were then tested in murine MC38 colon cancer.ResultsMouse ASD141 as monotherapy results in statistically significant growth inhibition in murine colon cancer models. Xi2396 remodels the TME by decreasing infiltration of TAMCs, and increased infiltration of dendritic cells (cDCs, NKDCs, and pDCs). Furthermore, Xi2396 also enhanced the antigen presentation ability, which is accompanied by an increased expression of MHCII, CD80 and CD86. These results indicate that the anti-CD11b monoclonal antibody, ASD141, designed to modulate TAMCs of the TME represents a novel approach of cancer immunotherapy.Xi2396 treatment also induced high levels of PD-L1 expression in the TME. Since PD-L1 expression in the TME was associated with response to current immune checkpoint blockades, we sought to determine whether Xi2396 treatment is capable of enhancing anti-tumor response to anti-PD1 therapy. Our results showed that combination of Xi2396 and anti-PD1 synergistically suppressed tumor growth.ConclusionsAltogether, our results provide support for clinical efforts to evaluate ASD141 as an innate immune checkpoint drug, especially in combination with commercial immune checkpoint inhibitors.Ethics ApprovalThis study was approved by National Laboratory Animal Center‘s Institutional Animal Care and Use Committee; approval number NLAC-110-D-006-R2.

Reduced Follicular Regulatory T Cells in Spleen and Pancreatic Lymph Nodes of Patients With Type 1 Diabetes

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at-risk for T1D, multiple studies have analyzed and reported alterations in follicular helper T cells (Tfh) in presymptomatic AAb-positive subjects and patients with T1D. Yet, it is still not clear whether the regulatory counterpart of Tfh cells, represented by follicular regulatory T cells (Tfr), is similarly altered. To address this question, we performed analyses in peripheral blood, spleen and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb-negative and -positive subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed between T1D, AAb-negative and AAb-positive adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared to non-diabetic controls. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.

Reduced Follicular Regulatory T Cells in Spleen and Pancreatic Lymph Nodes of Patients With Type 1 Diabetes

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at-risk for T1D, multiple studies have analyzed and reported alterations in follicular helper T cells (Tfh) in presymptomatic AAb-positive subjects and patients with T1D. Yet, it is still not clear whether the regulatory counterpart of Tfh cells, represented by follicular regulatory T cells (Tfr), is similarly altered. To address this question, we performed analyses in peripheral blood, spleen and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb-negative and -positive subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed between T1D, AAb-negative and AAb-positive adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared to non-diabetic controls. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.

Reduced Follicular Regulatory T Cells in Spleen and Pancreatic Lymph Nodes of Patients With Type 1 Diabetes

In the attempt to understand the origin of autoantibody (AAb) production in patients with and at-risk for T1D, multiple studies have analyzed and reported alterations in follicular helper T cells (Tfh) in presymptomatic AAb-positive subjects and patients with T1D. Yet, it is still not clear whether the regulatory counterpart of Tfh cells, represented by follicular regulatory T cells (Tfr), is similarly altered. To address this question, we performed analyses in peripheral blood, spleen and pancreatic lymph nodes (PLN) of organ donor subjects with T1D. Blood analyses were also performed in living AAb-negative and -positive subjects. While negligible differences in the frequency and phenotype of blood Tfr cells were observed between T1D, AAb-negative and AAb-positive adult subjects, the frequency of Tfr cells was significantly reduced in spleen and PLN of T1D as compared to non-diabetic controls. Furthermore, adoptive transfer of Tfr cells delayed disease development in a mouse model of T1D, a finding that could indicate that Tfr cells play an important role in peripheral tolerance and regulation of autoreactive Tfh cells. Together, our findings provide evidence of Tfr cell alterations within disease-relevant tissues in patients with T1D suggesting a role for Tfr cells in defective humoral tolerance and disease pathogenesis.