Differential effects of anti-CD20 therapy on CD4 and CD8 T cells and implication of CD20-expressing CD8 T cells in MS disease activity.

A small proportion of multiple sclerosis (MS) patients develop new disease activity soon after starting anti-CD20 therapy. This activity does not recur with further dosing, possibly reflecting deeper depletion of CD20-expressing cells with repeat infusions. We assessed cellular immune profiles and their association with transient disease activity following anti-CD20 initiation as a window into relapsing disease biology. Peripheral blood mononuclear cells from independent discovery and validation cohorts of MS patients initiating ocrelizumab were assessed for phenotypic and functional profiles using multiparametric flow cytometry. Pretreatment CD20-expressing T cells, especially CD20dimCD8+ T cells with a highly inflammatory and central nervous system (CNS)-homing phenotype, were significantly inversely correlated with pretreatment MRI gadolinium-lesion counts, and also predictive of early disease activity observed after anti-CD20 initiation. Direct removal of pretreatment proinflammatory CD20dimCD8+ T cells had a greater contribution to treatment-associated changes in the CD8+ T cell pool than was the case for CD4+ T cells. Early disease activity following anti-CD20 initiation was not associated with reconstituting CD20dimCD8+ T cells, which were less proinflammatory compared with pretreatment. Similarly, this disease activity did not correlate with early reconstituting B cells, which were predominantly transitional CD19+CD24highCD38high with a more anti-inflammatory profile. We provide insights into the mode-of-action of anti-CD20 and highlight a potential role for CD20dimCD8+ T cells in MS relapse biology; their strong inverse correlation with both pretreatment and early posttreatment disease activity suggests that CD20-expressing CD8+ T cells leaving the circulation (possibly to the CNS) play a particularly early role in the immune cascades involved in relapse development.

An antigen-encapsulating nanoparticle platform for TH1/17 immune tolerance therapy.

Tolerogenic nanoparticles (NPs) are rapidly being developed as specific immunotherapies to treat autoimmune disease. However, many NP-based therapies conjugate antigen (Ag) directly to the NP posing safety concerns due to antibody binding or require the co-delivery of immunosuppressants to induce tolerance. Here, we developed Ag encapsulated NPs comprised of poly(lactide-co-glycolide) [PLG(Ag)] and investigated the mechanism of action for Ag-specific tolerance induction in an autoimmune model of T helper type 1/17 dysfunction - relapse-remitting experimental autoimmune encephalomyelitis (R-EAE). PLG(Ag) completely abrogated disease induction in an organ specific manner, where the spleen was dispensable for tolerance induction. PLG(Ag) delivered intravenously distributed to the liver, associated with macrophages, and recruited Ag-specific T cells. Furthermore, programmed death ligand 1 (PD-L1) was increased on Ag presenting cells and PD-1 blockade lessened tolerance induction. The robust promotion of tolerance by PLG(Ag) without co-delivery of immunosuppressive drugs, suggests that these NPs effectively deliver antigen to endogenous tolerogenic pathways.

Cutting Edge: CD99 Is a Novel Therapeutic Target for Control of T Cell-Mediated Central Nervous System Autoimmune Disease.

Leukocyte trafficking into the CNS is a prominent feature driving the immunopathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Blocking the recruitment of inflammatory leukocytes into the CNS represents an exploitable therapeutic target; however, the adhesion molecules that specifically regulate the step of leukocyte diapedesis into the CNS remain poorly understood. We report that CD99 is critical for lymphocyte transmigration without affecting adhesion in a human blood-brain barrier model. CD99 blockade in vivo ameliorated experimental autoimmune encephalomyelitis and decreased the accumulation of CNS inflammatory infiltrates, including dendritic cells, B cells, and CD4(+) and CD8(+) T cells. Anti-CD99 therapy was effective when administered after the onset of disease symptoms and blocked relapse when administered therapeutically after disease symptoms had recurred. These findings underscore an important role for CD99 in the pathogenesis of CNS autoimmunity and suggest that it may serve as a novel therapeutic target for controlling neuroinflammation.

γδ T cell subsets play opposing roles in regulating experimental autoimmune encephalomyelitis.

γδ T cells are resident in cerebrospinal fluid and central nervous system (CNS) lesions of multiple sclerosis (MS) patients, but as multifaceted cells exhibiting innate and adaptive characteristics, their function remains unknown. Previous studies in experimental autoimmune encephalomyelitis (EAE) are contradictory and identified these cells as either promoting or suppressing disease pathogenesis. This study examines distinct γδ T cell subsets during EAE and indicates they mediate differential functions in CNS inflammation and demyelination resulting in pathogenesis or protection. We identified two γδ subsets in the CNS, Vγ1(+) and Vγ4(+), with distinct cytokine profiles and tissue specificity. Anti-γδ T cell receptor (TCR) monoclonal antibody (mAb) administration results in activation and downregulation of surface TCR, rendering the cells undetectable, but with opposing effects: anti-Vγ4 treatment exacerbates disease whereas anti-Vγ1 treatment is protective. The Vγ4(+) subset produces multiple pro-inflammatory cytokines including high levels of IL-17, and accounts for 15-20% of the interleukin-17 (IL-17) producing cells in the CNS, but utilize a variant transcriptional program than CD4(+) Th17 cells. In contrast, the Vγ1 subset produces CCR5 ligands, which may promote regulatory T cell differentiation. γδ T cell subsets thus play distinct and opposing roles during EAE, providing an explanation for previous reports and suggesting selective targeting to optimize regulation as a potential therapy for MS.

A biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease.

Targeted immune tolerance is a coveted therapy for the treatment of a variety of autoimmune diseases, as current treatment options often involve nonspecific immunosuppression. Intravenous (iv) infusion of apoptotic syngeneic splenocytes linked with peptide or protein autoantigens using ethylene carbodiimide (ECDI) has been demonstrated to be an effective method for inducing peripheral, antigen-specific tolerance for treatment of autoimmune disease. Here, we show the ability of biodegradable poly(lactic-co-glycolic acid) (PLG) nanoparticles to function as a safe, cost-effective, and highly efficient alternative to cellular carriers for the induction of antigen-specific T cell tolerance. We describe the formulation of tolerogenic PLG particles and demonstrate that administration of myelin antigen-coupled particles both prevented and treated relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a CD4 T cell-mediated mouse model of multiple sclerosis (MS). PLG particles made on-site with surfactant modifications surpass the efficacy of commercially available particles in their ability to couple peptide and to prevent disease induction. Most importantly, myelin antigen-coupled PLG nanoparticles are able to significantly ameliorate ongoing disease and subsequent relapses when administered at onset or at peak of acute disease, and minimize epitope spreading when administered during disease remission. Therapeutic treatment results in significantly reduced CNS infiltration of encephalitogenic Th1 (IFN-γ) and Th17 (IL-17a) cells as well as inflammatory monocytes/macrophages. Together, these data describe a platform for antigen display that is safe, low-cost, and highly effective at inducing antigen-specific T cell tolerance. The development of such a platform carries broad implications for the treatment of a variety of immune-mediated diseases.

The experimental autoimmune encephalomyelitis (EAE) model of MS: utility for understanding disease pathophysiology and treatment.

While no single model can exactly recapitulate all aspects of multiple sclerosis (MS), animal models are essential in understanding the induction and pathogenesis of the disease and to develop therapeutic strategies that limit disease progression and eventually lead to effective treatments for the human disease. Several different models of MS exist, but by far the best understood and most commonly used is the rodent model of experimental autoimmune encephalomyelitis (EAE). This model is typically induced by either active immunization with myelin-derived proteins or peptides in adjuvant or by passive transfer of activated myelin-specific CD4+ T lymphocytes. Mouse models are most frequently used because of the inbred genotype of laboratory mice, their rapid breeding capacity, the ease of genetic manipulation, and availability of transgenic and knockout mice to facilitate mechanistic studies. Although not all therapeutic strategies for MS have been developed in EAE, all of the current US Food and Drug Administration (FDA)-approved immunomodulatory drugs are effective to some degree in treating EAE, a strong indicator that EAE is an extremely useful model to study potential treatments for MS. Several therapies, such as glatiramer acetate (GA: Copaxone), and natalizumab (Tysabri), were tested first in the mouse model of EAE and then went on to clinical trials. Here we discuss the usefulness of the EAE model in understanding basic disease pathophysiology and developing treatments for MS as well as the potential drawbacks of this model.

High-mobility group box 1 protein (HMGB1) neutralization ameliorates experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease and as such, the gold standard of treatment is to selectively suppress the pathogenic autoimmune response without compromising the entire arm of the adaptive immune response. One target of this strategy lying upstream of the pathologic adaptive immune response is the local, innate immune signaling that initiates and drives autoimmunity and sterile injury. High-mobility group box 1 protein (HMGB1) is a ubiquitous nuclear protein that when released from necrotic cells, such as damaged oligodendrocytes in MS lesions, drives pro-inflammatory responses. Here we demonstrate that HMGB1 drives neuroinflammatory responses in experimental autoimmune encephalomyelitis (EAE), a murine model for MS, and that inhibition of HMGB1 signaling ameliorates disease. Specifically i.v. injection of an HMGB1 neutralizing antibody in the C57BL/6 model of chronic EAE or SJL/J model of relapsing-remitting EAE ameliorated clinical disease prophylactically or during ongoing disease, blocked T cell infiltration of the central nervous system, and inhibited systemic CD4(+) T cell responses to myelin epitopes. Additionally, lymphocytes from EAE mice restimulated in vitro in the presence of recombinant HMGB1 exhibited increased proliferation and pro-inflammatory cytokine production, an effect that was blocked by anti-HMGB1 antibody. Similarly recombinant HMGB1 promoted proliferation and pro-inflammatory cytokine production of human peripheral blood mononuclear cells stimulated in vitro, and anti-HMGB1 antibody blocked this effect. These findings indicate that HMGB1 contributes to neuroinflammatory responses that drive EAE pathogenesis and that HMGB1 blockade may be a novel means to selectively disrupt the pro-inflammatory loop that drives MS autoimmunity.

Antibody-independent B cell effector functions in relapsing remitting multiple sclerosis: clues to increased inflammatory and reduced regulatory B cell capacity.

The pathogenic role for B cells in the context of relapsing remitting multiple sclerosis (MS) is incompletely defined. Although classically considered a T cell-mediated disease, B cell-depleting therapies showed efficacy in treating the clinical symptoms of RRMS without decreasing plasma cells or total immunoglobulin (Ig) levels. Here, we discuss the potential implications of antibody-independent B cell effector functions that could contribute to autoimmunity with particular focus on antigen presentation, cytokine secretion, and stimulation of T cell subsets. We highlight differences between memory and naïve B cells from MS patients such as our recent findings of hyper-proliferation from MS memory B cells in response to CD40 engagement. We discuss the implications of IL6 overproduction in contrast to limited IL10 production by B cells from MS patients and comment on the impact of these functions on yet unexplored aspects of B cells in autoimmune disease. Finally, we contextualize B cell effector functions with respect to current immunomodulatory therapies for MS and show that glatiramer acetate (GA) does not directly modulate B cell proliferation or cytokine secretion.

Tolerance induced by apoptotic antigen-coupled leukocytes is induced by PD-L1+ and IL-10-producing splenic macrophages and maintained by T regulatory cells.

Ag-specific tolerance is a highly desired therapy for immune-mediated diseases. Intravenous infusion of protein/peptide Ags linked to syngeneic splenic leukocytes with ethylene carbodiimide (Ag-coupled splenocytes [Ag-SP]) has been demonstrated to be a highly efficient method for inducing peripheral, Ag-specific T cell tolerance for treatment of autoimmune disease. However, little is understood about the mechanisms underlying this therapy. In this study, we show that apoptotic Ag-SP accumulate in the splenic marginal zone, where their uptake by F4/80(+) macrophages induces production of IL-10, which upregulates the expression of the immunomodulatory costimulatory molecule PD-L1 that is essential for Ag-SP tolerance induction. Ag-SP infusion also induces T regulatory cells that are dispensable for tolerance induction but required for long-term tolerance maintenance. Collectively, these results indicate that Ag-SP tolerance recapitulates how tolerance is normally maintained in the hematopoietic compartment and highlight the interplay between the innate and adaptive immune systems in the induction of Ag-SP tolerance. To our knowledge, we show for the first time that tolerance results from the synergistic effects of two distinct mechanisms, PD-L1-dependent T cell-intrinsic unresponsiveness and the activation of T regulatory cells. These findings are particularly relevant as this tolerance protocol is currently being tested in a Phase I/IIa clinical trial in new-onset relapsing-remitting multiple sclerosis.

Rituximab therapy reduces organ-specific T cell responses and ameliorates experimental autoimmune encephalomyelitis.

Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.

Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein.

Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-α secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4(+) T-cell proliferation and IFN-γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4(+) T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells.

Potential of a unique antibody gene signature to predict conversion to clinically definite multiple sclerosis.

We identified a unique antibody gene mutation pattern (i.e. "signature") in cerebrospinal fluid (CSF) B cells from multiple sclerosis (MS) patients not present in control populations. Prevalence of the signature in CSF B cells of patients at risk to develop MS predicted conversion to MS with 91% accuracy in a small cohort of clinically isolated syndrome patients. If confirmed, signature prevalence would be a novel genetic diagnostic tool candidate for patients with early demyelinating disease of the central nervous system.

Impact of myelin-specific antigen presenting B cells on T cell activation in multiple sclerosis.

The role of B cells in the pathogenesis of Multiple Sclerosis (MS) is incompletely understood. Here we define a possible role for B cells as myelin-specific antigen presenting cells (B-APCs) in MS. Peripheral blood B cells (PBBC) isolated from both MS patients and healthy controls (HC) were activated in vitro with either CD40L/IL-4 or a Class B CpG oligodeoxynucleotide (CpG ODN)/IL-2. Both activation techniques induced PBBCs to upregulate CD80 and HLA-DR, rendering them more efficient APCs than resting B cells. Although the CD40L/IL-4 B-APCs were highly effective in eliciting CNS-antigen specific proliferation by autologous T cells, CpG ODN/IL-2 stimulated B cells were not. Furthermore, CD40L/IL-4 B-APC induced responses by autologous CD4(+) T cells were susceptible to blocking with anti-HLA-DR antibody, suggesting that T cell responses were specific for antigen presentation by B-APC. CNS-antigen specific CD8(+) T cell proliferation was also blocked by HLA-DR, suggesting that CD8(+) proliferation is in part dependent on CD4(+) help.