Proteolipid Protein-Induced Mouse Model of Multiple Sclerosis Requires B Cell-Mediated Antigen Presentation.

The pathogenic role B cells play in multiple sclerosis is underscored by the success of B cell depletion therapies. Yet, it remains unclear how B cells contribute to disease, although it is increasingly accepted that mechanisms beyond Ab production are involved. Better understanding of pathogenic interactions between B cells and autoreactive CD4 T cells will be critical for novel therapeutics. To focus the investigation on B cell:CD4 T cell interactions in vivo and in vitro, we previously developed a B cell-dependent, Ab-independent experimental autoimmune encephalomyelitis (EAE) mouse model driven by a peptide encompassing the extracellular domains of myelin proteolipid protein (PLPECD). In this study, we demonstrate that B cell depletion significantly inhibited PLPECD-induced EAE disease, blunted PLPECD-elicited delayed-type hypersensitivity reactions in vivo, and reduced CD4 T cell activation, proliferation, and proinflammatory cytokine production. Further, PLPECD-reactive CD4 T cells sourced from B cell-depleted donor mice failed to transfer EAE to naive recipients. Importantly, we identified B cell-mediated Ag presentation as the critical mechanism explaining B cell dependence in PLPECD-induced EAE, where bone marrow chimeric mice harboring a B cell-restricted MHC class II deficiency failed to develop EAE. B cells were ultimately observed to restimulate significantly higher Ag-specific proliferation from PLP178-191-reactive CD4 T cells compared with dendritic cells when provided PLPECD peptide in head-to-head cultures. We therefore conclude that PLPECD-induced EAE features a required pathogenic B cell-mediated Ag presentation function, providing for investigable B cell:CD4 T cell interactions in the context of autoimmune demyelinating disease.

A Functionally Distinct CXCR3+/IFN-γ+/IL-10+ Subset Defines Disease-Suppressive Myelin-Specific CD8 T Cells.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. We have previously demonstrated that CNS-specific CD8 T cells possess a disease-suppressive function in MS and variations of its animal model, experimental autoimmune encephalomyelitis (EAE), including the highly clinically relevant relapsing-remitting EAE disease course. Regulatory CD8 T cell subsets have been identified in EAE and other autoimmune diseases, but studies vary in defining phenotypic properties of these cells. In relapsing-remitting EAE, PLP178-191 CD8 T cells suppress disease, whereas PLP139-151 CD8 T cells lack this function. In this study, we used this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP178-191 CD8 T cells versus nonregulatory PLP139-151 or OVA323-339 CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3+ subpopulation among activated regulatory CD8 T cells, relative to nonregulatory counterparts. This subset exhibited increased degranulation and IFN-γ and IL-10 coproduction. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP178-191 CD8 T cells but not within nonregulatory OVA323-339 CD8 T cells. This disease-suppressing CD8 T cell subpopulation provides better insights into functional regulatory mechanisms, and targeted enhancement of this subset could represent a novel immunotherapeutic approach for MS.

Immune Autoregulatory CD8 T Cells Require IFN-γ Responsiveness to Optimally Suppress Central Nervous System Autoimmunity.

Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) and for developing successful immunotherapies. Our group has demonstrated that CNS myelin-specific CD8 T cells unexpectedly harbor immune regulatory capacity in both mouse and human. In particular, PLP178-191-specific CD8 T cells (PLP-CD8) robustly suppress the MS mouse model experimental autoimmune encephalomyelitis. We have recently shown that this depends on PLP-CD8 elaborating IFN-γ and perforin in a coordinated suppression program over time. However, the cellular target and downstream effects of CD8 T cell-derived IFN-γ remains poorly understood. In this study, we show that although wild-type (WT) PLP-CD8 were robustly suppressive in IFN-γR-deficient mice, IFN-γR-deficient PLP-CD8 exhibited suboptimal suppression in WT mice. Compared with WT counterparts, IFN-γR-deficient PLP-CD8 were defective in suppressing disease in IFN-γ-deficient recipients, a scenario in which the only IFN-γ available to WT PLP-CD8 is that which they produce themselves. Further, we found that IFN-γR-deficient PLP-CD8 exhibited altered granzyme/IFN-γ profiles, altered migration in recipients, and deficits in killing capacity in vivo. Collectively, this work suggests that IFN-γ responsiveness allows myelin-specific CD8 T cells to optimally perform autoregulatory function in vivo. These insights may help elucidate future adoptive immunotherapeutic approaches for MS patients.

Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

T regulatory cells participate in the control of germinal centre reactions.

Germinal centre (GC) reactions are central features of T-cell-driven B-cell responses, and the site where antibody-producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of high affinity antibodies. These processes require exquisite regulation not only to ensure the production of desired antibodies, but to minimize unwanted autoreactive or low affinity antibodies. To assess whether T regulatory (Treg) cells participate in the control of GC responses, immunized mice were treated with an anti-glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) monoclonal antibody (mAb) to disrupt Treg-cell activity. In anti-GITR-treated mice, the GC B-cell pool was significantly larger compared with control-treated animals, with switched GC B cells composing an abnormally high proportion of the response. Dysregulated GCs were also observed regardless of strain, T helper type 1 or 2 polarizing antigens, and were also seen after anti-CD25 mAb treatment. Within the spleens of immunized mice, CXCR5(+) and CCR7(-) Treg cells were documented by flow cytometry and Foxp3(+) cells were found within GCs using immunohistology. Final studies demonstrated administration of either anti-transforming growth factor-ß or anti-interleukin-10 receptor blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Treg cells is important in controlling the GC response. Taken together, these findings indicate that Treg cells contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs.

Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein.

Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the pathogenic and regulatory roles these cells play in demyelinating disease. Dissecting the function of B cells in the MS mouse model experimental autoimmune encephalomyelitis (EAE) is largely confined to induction with either the myelin oligodendrocyte glycoprotein epitope MOG35-55 or the full-length recombinant human MOG protein, the latter representing the most-used B cell-dependent EAE model. There is a clear need to investigate B cell function in additional myelin antigen contexts. Unlike MOG35-55, where lack of B cells yields more severe disease, we show here that the immunodominant myelin proteolipid protein epitope (PLP178-191) elicited identical EAE in WT and µMT mice, suggesting an absence of B cell engagement by this peptide. We hypothesized that a longer PLP antigen may better engage B cells and designed a peptide encompassing the extracellular domains (ECD) of PLP. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE. In contrast, PLPECD induced EAE not only in WT mice, but in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.

Therapeutic intervention in relapsing autoimmune demyelinating disease through induction of myelin-specific regulatory CD8 T cell responses.

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). We have shown that CNS-specific CD8 T cells (CNS-CD8) possess a disease suppressive function in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Previous studies have focused on the role of these cells predominantly in chronic models of disease, but the majority of MS patients present with a relapsing-remitting disease course. In this study, we evaluated the therapeutic role of CD8 T cells in the context of relapsing-remitting disease (RR-EAE), using SJL mice. We found that PLP178-191- and MBP84-104-CD8 ameliorated disease severity in an antigen-specific manner. In contrast, PLP139-151-CD8 did not suppress disease. PLP178-191-CD8 were able to reduce the number of relapses even when transferred during ongoing disease. We further ascertained that the suppressive subset of CD8 T cells was contained within the CD25+ CD8 T cell compartment post-in vitro activation with PLP178-191. Using Listeria monocytogenes (LM) encoding CNS antigens to preferentially prime suppressive CDS T cells in vivo, we show that LM infection induced disease suppressive CD8 T cells that protected and treated PLP178-191 disease. Importantly, a combination of PLP178-191-CDs transfer boosted by LM-PLP175-194 infection effectively treated ongoing disease induced by a non-cognate peptide (PLP139-151), indicating that this approach could be effective even in the context of epitope spreading. These data support a potential immunotherapeutic strategy using CD8 transfer and/or LM vaccination to boost disease regulatory CD8 T cells.

Early IFNγ-Mediated and Late Perforin-Mediated Suppression of Pathogenic CD4 T Cell Responses Are Both Required for Inhibition of Demyelinating Disease by CNS-Specific Autoregulatory CD8 T Cells.

Pathogenesis of immune-mediated demyelinating diseases like multiple sclerosis (MS) is thought to be governed by a complex cellular interplay between immunopathogenic and immunoregulatory responses. We have previously shown that central nervous system (CNS)-specific CD8 T cells have an unexpected protective role in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). In this study, we interrogated the suppressive potential of PLP178-191-specific CD8 T cells (PLP-CD8). Here, we show that PLP-CD8, when administered post-disease onset, rapidly ameliorated EAE progression, and suppressed PLP178-191-specific CD4 T cell responses as measured by delayed-type hypersensitivity (DTH). To accomplish DTH suppression, PLP-CD8 required differential production of perforin and IFNγ. Perforin was not required for the rapid suppressive action of these cells, but was critical for maintenance of optimal longer term DTH suppression. Conversely, IFNγ production by PLP-CD8 was necessary for swift DTH suppression, but was less significant for maintenance of longer term suppression. These data indicate that CNS-specific CD8 T cells employ an ordered regulatory mechanism program over a number of days in vivo during demyelinating disease and have mechanistic implications for this immunotherapeutic approach.

A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for multiple sclerosis and is characterized by infiltration of mononuclear cells and demyelination within the central nervous system along with the clinical symptoms of paralysis. EAE is a multifocal and random disease, which sometimes makes histopathologic analysis of lesions difficult as it may not be possible to predict where lesions will occur, especially when evaluating cross sections of spinal cord. Consequently, lesions may be easily missed due to limited sampling in traditional approaches. To evaluate the entire length of the spinal cord while maintaining anatomic integrity, we have developed a method to section the cord within the decalcified spinal column, which allows for the study of the multifocal nature of this disease and also minimizes handling artifact. HE and Luxol fast blue staining of these spinal cord sections revealed a paucity of lesions in some areas, while others showed marked inflammation and demyelination. The percentage of spinal cord affected by EAE was evaluated at four separate areas of longitudinally sectioned cord and it varied greatly within each animal. Immunohistochemical staining of in situ spinal cords which had undergone decalcification was successful for key immuno-markers used in EAE research including CD3 for T cells, B220 for B cells and F4/80 for murine macrophages. This method will allow investigators to look at the entire spinal cord on a single slide and evaluate the spinal cord with and without classic EAE lesions.

CD8(+) T-Cells as Immune Regulators of Multiple Sclerosis.

The vast majority of studies regarding the immune basis of MS (and its animal model, EAE) have largely focused on CD4(+) T-cells as mediators and regulators of disease. Interestingly, CD8(+) T-cells represent the predominant T-cell population in human MS lesions and are oligoclonally expanded at the site of pathology. However, their role in the autoimmune pathologic process has been both understudied and controversial. Several animal models and MS patient studies support a pathogenic role for CNS-specific CD8(+) T-cells, whereas we and others have demonstrated a regulatory role for these cells in disease. In this review, we describe studies that have investigated the role of CD8(+) T-cells in MS and EAE, presenting evidence for both pathogenic and regulatory functions. In our studies, we have shown that cytotoxic/suppressor CD8(+) T-cells are CNS antigen-specific, MHC class I-restricted, IFNγ- and perforin-dependent, and are able to inhibit disease. The clinical relevance for CD8(+) T-cell suppressive function is best described by a lack of their function during MS relapse, and importantly, restoration of their suppressive function during quiescence. Furthermore, CD8(+) T-cells with immunosuppressive functions can be therapeutically induced in MS patients by glatiramer acetate (GA) treatment. Unlike CNS-specific CD8(+) T-cells, these immunosuppressive GA-induced CD8(+) T-cells appear to be HLA-E restricted. These studies have provided greater fundamental insight into the role of autoreactive as well as therapeutically induced CD8(+) T-cells in disease amelioration. The clinical implications for these findings are immense and we propose that this natural process can be harnessed toward the development of an effective immunotherapeutic strategy.

Primary and long-term B-cell responses in the upper airway and lung after influenza A virus infection.

Influenza A virus (IAV) infection represents a significant global public health burden in addition to its potential as a pandemic killer. Accordingly, the immune response within the respiratory tract and associated lymphoid tissues has been a focus of study for decades. Murine model systems have led to a relatively clear understanding that while innate and T-cell responses are essential for clearance of an initial infection, high affinity neutralizing antibodies (Abs) generated by long-lived Ab forming cells and memory B cells are critical for protection from reinfection and are the goal of classic vaccination strategies. Indeed, the local and systemic IAV-specific Ab response after primary pulmonary infection has been well studied in mice. However, the highly organized microenvironments responsible for producing long-lived, high affinity responses, namely germinal center (GC) reactions, have been less well studied. Recently, work from our laboratory and others has provided new insights into the induction and character of IAV-specific GC responses in the secondary lymphoid organs and the lung. Of interest, these studies have demonstrated IAV reactive GCs to persist for extended periods in both draining lymph nodes and lung tissue. Herein, the primary adaptive response to IAV is reviewed with an emphasis on GC B-cell responses. In addition, data are shown demonstrating the persistence of GCs in the respiratory tract after IAV infection, and key factors that drive their maintenance.

Pulmonary infection with influenza A virus induces site-specific germinal center and T follicular helper cell responses.

Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These B cell subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the GC reaction after respiratory IAV infection is lacking, as is the characterization of T follicular helper (T(FH)) cells that support GCs. Here, GC B cell and T(FH) cell responses were studied in mice following pulmonary challenge with IAV. Marked GC reactions were induced in draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude and kinetics of the response was site-specific. Examination of switching within GCs demonstrated IgG2(+) cells to compose the largest fraction in dLNs, lung and spleen. IgA(+) GC B cells were infrequent in these sites, but composed a significant subset of the switched GC population in NALT. Further experiments demonstrated splenectomized mice to withstand a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection in spite of strong GC responses in this organ. Final studies showed that T(FH) cell numbers were highest in dLNs and spleen, and peaked in all sites prior to the height of the GC reaction. T(FH) cells purified from dLNs generated IL-21 and IFNγ upon activation, although CD4(+)CXCR5(-) T effector cells produced higher levels of all cytokines. Collectively, these findings reveal respiratory IAV infection to induce strong T helper cell-driven B cell responses in various organs, with each site displaying unique attributes.