AMD3100-Mediated CXCR4 Inhibition Impairs Development of Primary Lymphoma of the Central Nervous System.

A hallmark of primary lymphoma of the central nervous system (CNS; PCNSL) is the strong CXCR4 expression of the tumor cells, the function of which is still unknown. In vitro treatment of BAL17CNS lymphoma cells by AMD3100, which inhibits CXCR4-CXCL12 interactions, resulted in the significantly differential expression of 273 genes encoding proteins involved in cell motility, cell-cell signaling and interaction, hematological system development and function, and immunologic disease. Among the genes down-regulated was the one encoding CD200, a regulator of CNS immunologic activity. These data directly translated into the in vivo situation; BAL17CNS CD200 expression was down-regulated by 89% (3% versus 28% CD200+ lymphoma cells) in AMD3100-treated versus untreated mice with BAL17CNS-induced PCNSL. Reduced lymphoma cell CD200 expression may contribute to the markedly increased microglial activation in AMD3100-treated mice. AMD3100 also maintained the structural integrity of blood-brain barrier tight junctions and the outer basal lamina of cerebral blood vessels. Subsequently, lymphoma cell invasion of the brain parenchyma was impaired, and maximal parenchymal tumor size was significantly reduced by 82% in the induction phase. Thus, AMD3100 qualified as a potentially attractive candidate to be included into the therapeutic concept of PCNSL. Beyond therapy, CXCR4-induced suppression of microglial activity is of general neuroimmunologic interest. This study identified CD200 expressed by the lymphoma cells as a novel mechanism of immune escape in PCNSL.

Expression of Cas9 in a Syngeneic Model of Primary Central Nervous System Lymphoma Induces Intracerebral NK and CD8 T Cell-Mediated Lymphoma Cell Lysis Via Perforin.

The development of clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene modification has opened an exciting avenue of targeting genes to study the pathogenesis of diseases and to develop novel therapeutic concepts. However, as the effector protein Cas9 is of bacterial origin, unwanted side effects due to a host immune response against Cas9 need to be considered. Here, we used the syngeneic model of BAL17CNS-induced primary lymphoma of the central nervous system (PCNSL, CNS) in BALB/c mice to address this issue. Surprisingly, stable expression of Cas9 in BAL17CNS (BAL17CNS/Cas9) cells rendered them unable to establish PCNSL on intracerebral transplantation. Instead, they induced a prominent intracerebral immune response mediated by CD8 T cells, which lysed BAL17CNS/Cas9 cells via perforin. In addition, B cells contributed to the immune response as evidenced by serum anti-Cas9 antibodies in BALB/c mice as early as day 8 after transplantation of BAL17CNS/Cas9 cells. In athymic BALB/cnu/nu mice, NK cells mounted a vigorous intracerebral immune response with perforin-mediated destruction of BAL17CNS/Cas9 cells. Thus, in the CNS, perforin produced by NK and CD8 T cells was identified as a mediator of cytotoxicity against BAL17CNS/Cas9 cells. These observations should be taken into account when considering therapeutic CRISPR-Cas9-mediated tumor cell manipulation for PCNSL.

Impact of a Faulty Germinal Center Reaction on the Pathogenesis of Primary Diffuse Large B Cell Lymphoma of the Central Nervous System.

Primary lymphoma of the central nervous system (PCNSL, CNS) is a specific diffuse large B cell lymphoma (DLBCL) entity confined to the CNS. Key to its pathogenesis is a failure of B cell differentiation and a lack of appropriate control at differentiation stages before entrance and within the germinal center (GC). Self-/polyreactive B cells rescued from apoptosis by MYD88 and/or CD79B mutations accumulate a high load of somatic mutations in their rearranged immunoglobulin (IG) genes, with ongoing somatic hypermutation (SHM). Furthermore, the targeting of oncogenes by aberrant SHM (e.g., PIM1, PAX5, RHOH, MYC, BTG2, KLHL14, SUSD2), translocations of the IG and BCL6 genes, and genomic instability (e.g., gains of 18q21; losses of 9p21, 8q12, 6q21) occur in these cells in the course of their malignant transformation. Activated Toll-like receptor, B cell receptor (BCR), and NF-κB signaling pathways foster lymphoma cell proliferation. Hence, tumor cells are arrested in a late B cell differentiation stage, corresponding to late GC exit B cells, which are genetically related to IgM+ memory cells. Paradoxically, the GC reaction increases self-/polyreactivity, yielding increased tumor BCR reactivity for multiple CNS proteins, which likely contributes to CNS tropism of the lymphoma. The loss of MHC class I antigen expression supports tumor cell immune escape. Thus, specific and unique interactions of the tumor cells with resident CNS cells determine the hallmarks of PCNSL.

CD8 T cell-Derived Perforin and TNF-α Are Crucial Mediators of Neuronal Destruction in Experimental Autoimmune Enteric Ganglionitis.

In neuron-specific ovalbumin-transgenic CKTAC mice, antigen-specific OT-I CD8 T cells home to the enteric nervous system, where they attack and destroy neurons of the myenteric and submucosal plexus. Clinically, experimental autoimmune enteric ganglionitis (EAEG) manifests with gastrointestinal dysmotility and rapidly progresses to lethal ileus. Although interferon-γ has been identified as capable of damaging neurons in EAEG, the role of perforin, Fas/FasL, and tumor necrosis factor-α (TNF-α) in this disease is still a matter of debate. Thus, CKTAC mice were adoptively transferred with either perforin-/- or wild-type OT-I CD8 T cells. In addition, CKTAC mice that had received wild-type OT-I CD8 T cells were treated by either anti-TNF-α or anti-FasL. Furthermore, wild-type OT-I CD8 T cells were adoptively transferred into CKTAC mice with neuron-specific deletion of Fas. Although neither inactivation of enteric neuronal Fas nor anti-FasL treatment improved the disease, the absence of perforin from OT-I CD8 T cells and anti-TNF-α treatment significantly ameliorated EAEG and prevented lethal ileus by rescue of enteric neurons. Thus, these experiments identify perforin and TNF-α as important in the pathogenesis of EAEG.

TLR signals license CD8 T cells to destroy oligodendrocytes expressing an antigen shared with a Listeria pathogen.

Increasing evidence suggests a role of CD8 T cells in autoimmune demyelinating CNS disease, which, however, is still controversially discussed. Mice, which express ovalbumin (OVA) as cytosolic self-antigen in oligodendrocytes (ODC-OVA mice), respond to CNS infection induced by OVA-expressing attenuated Listeria with CD8 T cell-mediated inflammatory demyelination. This model is suitable to decipher the contribution of CD8 T cells and the pathogen in autoimmune CNS disease. Here, we show that both antigen and pathogen are required in the CNS for disease induction, though not in a physically linked fashion. Intracerebral challenge with combined toll like receptor (TLR) TLR2 and TLR9 as well as TLR7 and TLR9 agonists substituted for the bacterial stimulus, but not with individual TLR agonists (TLR2, TLR3,TLR5,TLR7, TLR9). Furthermore, MyD88 inactivation rendered ODC-OVA mice resistant to disease induction. Collectively, CD8 T cell-mediated destruction of oligodendrocytes is activated if (i) an antigen shared with an infectious agent is provided in the CNS microenvironment and (ii) innate immune signals inform the CNS microenvironment that pathogen removal warrants an immune attack by CD8 T cells, even at the expense of locally restricted demyelination.

Enteric Murine Ganglionitis Induced by Autoimmune CD8 T Cells Mimics Human Gastrointestinal Dysmotility.

Inflammatory bowel diseases frequently cause gastrointestinal dysmotility, suggesting that they may also affect the enteric nervous system. So far, the precise mechanisms that lead to gastrointestinal dysmotility in inflammatory bowel diseases have not been elucidated. To determine the effect of CD8 T cells on gastrointestinal motility, transgenic mice expressing ovalbumin on enteric neurons were generated. In these mice, adoptive transfer of ovalbumin-specific OT-I CD8 T cells induced severe enteric ganglionitis. CD8 T cells homed to submucosal and myenteric plexus neurons, 60% of which were lost, clinically resulting in severely impaired gastrointestinal transition. Anti-interferon-γ treatment rescued neurons by preventing their up-regulation of major histocompatibility complex class I antigen, thus preserving gut motility. These preclinical murine data translated well into human gastrointestinal dysmotility. In a series of 30 colonic biopsy specimens from patients with gastrointestinal dysmotility, CD8 T cell-mediated ganglionitis was detected that was followed by severe loss of enteric neurons (74.8%). Together, the preclinical and clinical data support the concept that autoimmune CD8 T cells play an important pathogenetic role in gastrointestinal dysmotility and may destroy enteric neurons.

Mechanisms of intracerebral lymphoma growth delineated in a syngeneic mouse model of central nervous system lymphoma.

Primary lymphoma of the central nervous system (PCNSL) is defined as lymphoma of the diffuse large B-cell type confined to the CNS. To understand the effects of the CNS microenvironment on the malignant B cells and their interactions with the cells of the target organ, we analyzed a syngeneic mouse model. Transplantation of BAL17 cells into the frontal white matter of syngeneic BALB/c mice induced lymphomas with major clinical and neuropathologic features that parallel those of human PCNSL, including an angiocentric growth pattern in the brain parenchyma and tropism for the inner and outer ventricular system. Seven cycles of repeated isolation of lymphoma cells from the CNS and their intracerebral reimplantation induced genotypic and phenotypic alterations in resulting BAL17VII cells; the affected genes regulate apoptosis and are of the JAK/STAT pathway. Because lymphoma growth of BAL17VII cells was significantly accelerated, that is, shortening the time to death of the mice, these data indicate that prolonged stay of the lymphoma cells in the CNS was associated with worse outcome. These findings suggest that the CNS microenvironment fosters aggressiveness of lymphoma cells, thereby accelerating the lethal course of PCNSL.

Oligodendrocytes enforce immune tolerance of the uninfected brain by purging the peripheral repertoire of autoreactive CD8+ T cells.

Myelin-specific CD8(+) T cells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) T cells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) T cells failed to induce disease and were cleared from the immune system after antigen encounter in the central nervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) T cells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) T cells to cause demyelinating disease. Thus, in response to infection, CD8(+) T cells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

Role of CD8 T-cell-mediated autoimmune diseases of the central nervous system.

In T-cell-mediated autoimmune diseases of the central nervous system (CNS), CD4 T cells have long been regarded as the only pathogenetically relevant T-cell population. However, growing clinical and experimental evidence suggests that CD8 T cells also contribute significantly to autoimmune responses in the CNS. We discuss the potential induction of autoimmune CD8 T cells by infections, the impact of the microenvironment of the CNS on CD8 T-cell responses, and the potential interaction of CD8 T cells with autoantigen-expressing resident brain-cell populationsneurons in particularin light of clinical and experimental findings.

Dual role of B cells with accelerated onset but reduced disease activity in P0106₋125-induced experimental autoimmune neuritis of IgH °(/)° mice.

The role of B cells in autoimmune-mediated diseases of the peripheral nervous system was studied in experimental autoimmune neuritis (EAN) in B cell deficient IgH°(/)° C57BL/6J mice having been immunized with P0106₋125 peptide. Compared to coisogenic IgH(+/+) mice, onset of EAN was accelerated [100% disease incidence at day 9 post immunization (p.i.) vs. day 15 p.i.]. At day 9 p.i., numbers of P0106₋125-specific interferon (IFN)-γ-producing CD4(+) T cells were increased, while IL-10 mRNA and production were decreased in IgH°(/)° mice. Beyond day 9 p.i., declining disease activity and a significant reduction of maximal disease activity were correlated with significantly reduced numbers of IFN-γ-producing CD4(+) T cells in IgH(0/0) mice as compared with IgH(+/+) mice. Correspondingly, neuropathology demonstrated only mild axonal damage, while demyelination and dying back axonopathy with spinal cord motor neuron apoptosis were absent. Thus, depending on the stage of EAN, B cells play a dual, i.e. suppressive and enhancing, role during induction and at height of EAN, respectively. The combined interaction of B cells as well as CD4(+) and CD8(+) T cells is required for the development of EAN.

Molecular mimicry between neurons and an intracerebral pathogen induces a CD8 T cell-mediated autoimmune disease.

To identify basic mechanisms of how infections may induce a neuron-specific autoimmune response, we generated mice expressing OVA as neuronal autoantigen under control of the neuron-specific enolase promoter (NSE-OVA mice). Intracerebral, but not systemic, infection with attenuated Listeria monocytogenes-secreting OVA induced an atactic-paretic neurological syndrome in NSE-OVA mice after bacterial clearance from the brain, whereas wild-type mice remained healthy. Immunization with attenuated Listeria monocytogenes-secreting OVA before intracerebral infection strongly increased the number of intracerebral OVA-specific CD8 T cells aggravating neurological disease. T cell depletion and adoptive transfer experiments identified CD8 T cells as decisive mediators of the autoimmune disease. Importantly, NSE-OVA mice having received OVA-specific TCR transgenic CD8 T cells developed an accelerated, more severe, and extended neurological disease. Adoptively transferred pathogenic CD8 T cells specifically homed to OVA-expressing MHC class I(+) neurons and, corresponding to the clinical symptoms, approximately 30% of neurons in the anterior horn of the spinal cord became apoptotic. Thus, molecular mimicry between a pathogen and neurons can induce a CD8 T cell-mediated neurological disease, with its severity being influenced by the frequency of specific CD8 T cells, and its induction, but not its symptomatic phase, requiring the intracerebral presence of the pathogen.

Both TLR2 and TLR4 are required for the effective immune response in Staphylococcus aureus-induced experimental murine brain abscess.

Toll-like receptors (TLRs) play central roles in the innate reaction to bacterial products and transmit specific immune responses against these pathogens. TLRs are expressed on numerous cell types, including innate immune cells, and on astrocytes, neurons, and microglial cells of the central nervous system (CNS). Lipoproteins and lipopolysaccharides are specifically recognized by TLR2 and TLR4, respectively. We examined the in vivo role of TLR2 and TLR4 in Staphylococcus aureus-induced brain abscess. Phenotypically, 87% of TLR2(-/-) mice and 43% of TLR4(-/-) mice died whereas all wild-type (WT) mice recovered. Clearance of bacteria from the CNS was significantly delayed in TLR2(-/-) mice compared with TLR4(-/-) and WT animals. Recruitment of granulocytes and macrophages to the CNS, as well as microglial activation and expansion, was up-regulated in TLR2(-/-) mice. Although inflammation persisted especially in the CNS of TLR2(-/-) mice, but also of TLR4(-/-) mice, WT mice terminated the infection more effectively. Collectively, these data show that the immune response to experimental S. aureus-induced brain abscess depends crucially on the recognition of S. aureus by TLR2 but that TLR4 is also required for an optimal intracerebral immune response in this disorder.

Regulation of the inflammatory response to Staphylococcus aureus-induced brain abscess by interleukin-10.

A characteristic of brain abscess is a localized suppurative infection leading to substantial damage of the adjacent central nervous system tissue. The orchestrated interplay of pro- and antiinflammatory cytokines released by leukocytes as well as resident cells of the central nervous system is crucial for both an effective host defense and for limiting tissue damage in brain abscess. To study the regulatory role of interleukin (IL)-10 in brain abscess in vivo, IL-10-deficient (IL-10(0/0)) mice were stereotaxically infected with Staphylococcus aureus-laden agarose beads. Increased numbers of intracerebral (IC) granulocytes, macrophages, CD4+ and CD8+ T cells, and higher levels of TNF, IL-1beta, and iNOS were observed in IL-10(0/0) mice than in wild-type mice, whereas chemokines were induced earlier and more pronounced in wild-type mice. Together with prominent microvascular hemorrhage, necrotic vasculitis, severe brain edema, and markedly increased abscess size, these alterations led to an increased morbidity of IL-10(0/0) mice. Nevertheless, the hyperinflammatory response of IL-10(0/0) mice did not improve bacterial elimination. Collectively, these data outline the important role of IL-10 in vivo for the regulation of the IC host immune response in experimental S. aureus-induced brain abscess.