Jozsef Karman

Initiation of Immune Responses in Brain Is Promoted by Local Dendritic Cells

The contribution of dendritic cells (DCs) to initiating T cell-mediated immune response in and T cell homing into the CNS has not yet been clarified. In this study we show by confocal microscopy and flow cytometry that cells expressing CD11c, CD205, and MHC class II molecules and containing fluorescently labeled, processed Ag accumulate at the site of intracerebral Ag injection. These cells follow a specific pattern upon migrating out of the brain. To track their pathway out of the CNS, we differentiated DCs from bone marrow of GFP-transgenic mice and injected them directly into brains of naive C57BL/6 mice. We demonstrate that DCs migrate from brain to cervical lymph nodes, a process that can be blocked by fixation or pertussis toxin treatment of the DCs. Injection of OVA-loaded DCs into brain initiates a SIINFEKL (a dominant OVA epitope)-specific T cell response in lymph nodes and spleen, as measured by specific tetramer and LFA-1 activation marker staining. Additionally, a fraction of activated SIINFEKL-specific T cells home to the CNS. Specific T cell homing to the CNS, however, cannot be induced by i.v. injection of OVA-loaded DCs alone. These data suggest that brain-emigrant DCs are sufficient to support activated T cells to home to the tissue of DC origination. Thus, initiation of immune reactivity against CNS Ags involves the migration of APCs from nervous tissue to peripheral lymphoid tissues, similarly to that in other organs.

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1α increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1α-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.

Characterization of the Histoplasma capsulatum-Induced Granuloma

Rising rates of Histoplasma capsulatum infection are an emerging problem among the rapidly growing population of immune-compromised individuals. Although there is a growing understanding of systemic immunity against Histoplasma, little is known about the local granulomatous response, which is an important component in the control of infection. The focus of this article is the characterization of Histoplasma-induced granulomas. Five days after i.p. infection, infected macrophage appear in the liver and lung; however, no granulomas are apparent. Two days later, well-formed sarcoid granulomas are abundant in the lung and liver of infected mice, which contain all visible Histoplasma. Granulomas are dominated by macrophage and lymphocytes. Most of the Histoplasma and most of the apoptotic cells are found in the center of the lesions. We isolated liver granulomas at multiple time points after infection and analyzed the cellular composition, TCR gene usage, and cytokine production of granuloma-infiltrating cells. The lesions contain both CD4+ and CD8+ T cell subsets, and T cells are the primary source of IFN-γ and IL-17. The main source of local TNF-α is macrophage. Chemokines are produced by both infiltrating macrophage and lymphocytes. Dendritic cells are present in granulomas; however, T cell expansion seems to occur systemically because TCR usage is very heterogeneous even at the level of individual lesions. This study is the first direct examination of host cellular responses in the Histoplasma-induced granuloma representing the specific interface between host and pathogen. Our studies will allow further analysis of key elements of host Histoplasma interactions at the site of chronic infection.

Dendritic Cells Amplify T Cell-Mediated Immune Responses in the Central Nervous System

Neuroinflammation often starts with the invasion of T lymphocytes into the CNS leading to recruitment of macrophages and amplification of inflammation. In this study, we show that dendritic cells (DCs) facilitate T-T cell help in the CNS and contribute to the amplification of local neuroinflammation. We adoptively transferred defined amounts of naive TCR-transgenic (TCR) recombination-activating gene-1-deficient T cells into another TCR-transgenic mouse strain expressing different Ag specificity. Following adoptive transfers, we coinjected DCs that presented one or multiple Ags into the brain and followed the activation of T cells with defined specificities simultaneously. Injection of DCs presenting both Ags simultaneously led to significantly higher infiltration of T cells into the brain compared with injection of a mixture of DCs pulsed with two Ags separately. DCs mediated either cooperative or competitive interactions between T cell populations with different specificities depending upon their MHC-restricting element usage. These results suggest that DC-mediated cooperation between brain-infiltrating T cells of different Ag specificities in the CNS plays an important role in regulation of neuroinflammation. This work also implies that blocking Ag-specific responses may block not only the targeted specificities, but may also effectively block their cooperative assistance to other T cells. Therefore, these data justify more attention to Ag-specific therapeutic approaches for neuroinflammation.

Immune privilege of the CNS is not the consequence of limited antigen sampling

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Substance P receptor mediated maintenance of chronic inflammation in EAE.

Substance P (SP) is a modulatory, pro-inflammatory neuropeptide. We investigated the role of the SP receptor, neurokinin-1 (NK-1), in EAE. Our data show that in the chronic phase, mice lacking NK-1 have improved mobility and decreased numbers of LFA-1 high CD4+ T cells and MOG-specific, IFN-gamma producing CD4+ T cells. SR140333, an NK-1 antagonist, administered alone during the chronic phase of EAE was not sufficient to ameliorate symptoms. These results indicate that SP, through NK-1, contributes to maintenance of CNS inflammation, and combining NK-1 antagonists with conventional anti-inflammatory treatments may enhance the success of treatments for diseases like multiple sclerosis.

Interactions between T cells responding to concurrent mycobacterial and influenza infections.

CD4+ T cells are central in mediating granuloma formation and limiting growth and dissemination of mycobacterial infections. To determine whether T cells responding to influenza infection can interact with T cells responding to Mycobacterium bovis bacille Calmette-Guérin (BCG) infection and disrupt granuloma formation, we infected mice containing two monoclonal T cell populations specific for the model Ags pigeon cytochrome c (PCC) and hen egg lysozyme (HEL). These mice were chronically infected with PCC epitope-tagged BCG (PCC-BCG) and acutely infected with HEL epitope-tagged influenza virus (HEL-flu). In these mice, PCC-BCG infection is much more abundant in the liver than the lung, whereas HEL-flu infection is localized to the lung. We observe that both T cells have access to both inflammatory sites, but that PCC-specific T cells dominate the PCC-BCG inflammatory site in the liver, whereas HEL-specific T cells dominate the HEL-flu inflammatory site in the lung. Influenza infection, in the absence of an influenza-specific T cell response, is able to increase the activation state and IFN-γ secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-γ and contribute to protection in the granuloma. Ultimately, infection with influenza has little effect on bacterial load, and bacteria do not disseminate. In summary, these data illustrate complex interactions between T cell responses to infectious agents that can affect effector responses to pathogens.

Virally Activated CD8 T Cells Home to Mycobacterium bovis BCG-Induced Granulomas but Enhance Antimycobacterial Protection Only in Immunodeficient Mice

ABSTRACT The effect of secondary infections on CD4 T-cell-regulated chronic granulomatous inflammation is not well understood. Here, we have investigated the effect of an acute viral infection on the cellular composition and bacterial protection in Mycobacterium bovis strain bacille Calmette-Guérin (BCG)-induced granulomas using an immunocompetent and a partially immunodeficient murine model. Acute lymphocytic choriomeningitis virus (LCMV) coinfection of C57BL/6 mice led to substantial accumulation of gamma interferon (IFN-γ)-producing LCMV-specific T cells in liver granulomas and increased local IFN-γ. Despite traffic of activated T cells that resulted in a CD8 T-cell-dominated granuloma, the BCG liver organ load was unaltered from control levels. In OT-1 T-cell-receptor (TCR) transgenic mice, ovalbumin (OVA) immunization or LCMV coinfection of BCG-infected mice induced CD8 T-cell-dominated granulomas containing large numbers of non-BCG-specific activated T cells. The higher baseline BCG organ load in this CD8 TCR transgenic animal allowed us to demonstrate that OVA immunization and LCMV coinfection increased anti-BCG protection. The bacterial load remained substantially higher than in mice with a more complete TCR repertoire. Overall, the present study suggests that peripherally activated CD8 T cells can be recruited to chronic inflammatory sites, but their contribution to protective immunity is limited to conditions of underlying immunodeficiency.

Regional distribution of CNS antigens differentially determines T-cell mediated neuroinflammation in a CX3CR1 dependent manner

T cells continuously sample CNS-derived antigens in the periphery, yet it is unknown how they sample and respond to CNS antigens derived from distinct brain areas. We expressed ovalbumin (OVA) neoepitopes in regionally distinct CNS areas (Cnp-OVA and Nes-OVA mice) to test peripheral antigen sampling by OVA-specific T cells under homeostatic and neuroinflammatory conditions. We show that antigen sampling in the periphery is independent of regional origin of CNS antigens in both male and female mice. However, experimental autoimmune encephalomyelitis (EAE) is differentially influenced in Cnp-OVA and Nes-OVA female mice. Although there is the same frequency of CD45high CD11b+ CD11c+ CX3CL1+ myeloid cell–T-cell clusters in neoepitope-expressing areas, EAE is inhibited in Nes-OVA female mice and accelerated in CNP-OVA female mice. Accumulation of OVA-specific T cells and their immunomodulatory effects on EAE are CX3C chemokine receptor 1 (CX3CR1) dependent. These data show that despite similar levels of peripheral antigen sampling, CNS antigen-specific T cells differentially influence neuroinflammatory disease depending on the location of cognate antigens and the presence of CX3CL1/CX3CR1 signaling. SIGNIFICANCE STATEMENT Our data show that peripheral T cells similarly recognize neoepitopes independent of their origin within the CNS under homeostatic conditions. Contrastingly, during ongoing autoimmune neuroinflammation, neoepitope-specific T cells differentially influence clinical score and pathology based on the CNS regional location of the neoepitopes in a CX3CR1-dependent manner. Altogether, we propose a novel mechanism for how T cells respond to regionally distinct CNS derived antigens and contribute to CNS autoimmune pathology.