Julia Sim

BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1

During activation, T cells express receptors for receiving positive and negative costimulatory signals. Here we identify the B and T lymphocyte attenuator (BTLA), an immunoglobulin domain–containing glycoprotein with two immunoreceptor tyrosine-based inhibitory motifs. BTLA is not expressed by naive T cells, but it is induced during activation and remains expressed on T helper type 1 (TH1) but not TH2 cells. Crosslinking BTLA with antigen receptors induces its tyrosine phosphorylation and association with the Src homology domain 2 (SH2)-containing protein tyrosine phosphatases SHP-1 and SHP-2, and attenuates production of interleukin 2 (IL-2). BTLA-deficient T cells show increased proliferation, and BTLA-deficient mice have increased specific antibody responses and enhanced sensitivity to experimental autoimmune encephalomyelitis. B7x, a peripheral homolog of B7, is a ligand of BTLA. Thus, BTLA is a third inhibitory receptor on T lymphocytes with similarities to cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) and programmed death 1 (PD-1).

The AP-1 transcription factor Batf controls TH17 differentiation

Activator protein 1 (AP-1, also known as JUN) transcription factors are dimers of JUN, FOS, MAF and activating transcription factor (ATF) family proteins characterized by basic region and leucine zipper domains. Many AP-1 proteins contain defined transcriptional activation domains, but BATF and the closely related BATF3 (refs 2, 3) contain only a basic region and leucine zipper, and are considered to be inhibitors of AP-1 activity. Here we show that Batf is required for the differentiation of IL17-producing T helper (TH17) cells. TH17 cells comprise a CD4+ T-cell subset that coordinates inflammatory responses in host defence but is pathogenic in autoimmunity. Batf-/- mice have normal TH1 and TH2 differentiation, but show a defect in TH17 differentiation, and are resistant to experimental autoimmune encephalomyelitis. Batf-/- T cells fail to induce known factors required for TH17 differentiation, such as RORγt (encoded by Rorc) and the cytokine IL21 (refs 14–17). Neither the addition of IL21 nor the overexpression of RORγt fully restores IL17 production in Batf-/- T cells. The Il17 promoter is BATF-responsive, and after TH17 differentiation, BATF binds conserved intergenic elements in the Il17a–Il17f locus and to the Il17, Il21 and Il22 (ref. 18) promoters. These results demonstrate that the AP-1 protein BATF has a critical role in TH17 differentiation.

Regional CNS responses to IFN-γ determine lesion localization patterns during EAE pathogenesis

The localization of inflammatory foci within the cerebellum is correlated to severe clinical outcomes in multiple sclerosis (MS). Previous studies of experimental autoimmune encephalomyelitis (EAE), a model of MS, revealed distinct clinical outcomes correlated with the capacity of the animal to produce IFN-γ. Outcomes were linked to localization of inflammatory cells in either the spinal cord (wild type [WT]) or the cerebellum and brain stem (IFN-γ deficient). We demonstrate, using an adoptive transfer system, that the ability of the central nervous system (CNS) to sense pathogenic T cell–produced IFN-γ during EAE initiation determines the sites of CNS pathogenesis. Transfer of WT Th1 cells into IFN-γ receptor–deficient mice results in pathogenic invasion of the brain stem and cerebellum with attendant clinical symptoms, which are identical to the disease observed after transfer of IFN-γ–deficient T cells to WT hosts. Inflammation of the spinal cord associated with classical EAE is abrogated in both IFN-γ–deficient systems. Cotransfer of CNS antigen-specific WT Th1 cells with IFN-γ–deficient T cells is sufficient to restore spinal cord invasion and block cerebellar and brain stem invasion. These data demonstrate that interaction between IFN-γ and host CNS cells during the initiation of EAE can selectively promote or suppress neuroinflammation and pathogenesis.

Batf3-Dependent CD11blow/− Peripheral Dendritic Cells Are GM-CSF-Independent and Are Not Required for Th Cell Priming after Subcutaneous Immunization

Dendritic cells (DCs) subsets differ in precursor cell of origin, functional properties, requirements for growth factors, and dependence on transcription factors. Lymphoid-tissue resident CD8α+ conventional DCs (cDCs) and CD11blow/−CD103+ non-lymphoid DCs are developmentally related, each being dependent on FMS-like tyrosine kinase 3 ligand (Flt3L), and requiring the transcription factors Batf3, Irf8, and Id2 for development. It was recently suggested that granulocyte/macrophage colony stimulating factor (GM-CSF) was required for the development of dermal CD11blow/−Langerin+CD103+ DCs, and that this dermal DC subset was required for priming autoreactive T cells in experimental autoimmune encephalitis (EAE). Here, we compared development of peripheral tissue DCs and susceptibility to EAE in GM-CSF receptor deficient (Csf2rb −/−) and Batf3 −/− mice. We find that Batf3-dependent dermal CD11blow/−Langerin+ DCs do develop in Csf2rb −/− mice, but that they express reduced, but not absent, levels of CD103. Further, Batf3 −/− mice lacking all peripheral CD11blow/− DCs show robust Th cell priming after subcutaneous immunization and are susceptible to EAE. Our results suggest that defective T effector priming and resistance to EAE exhibited by Csf2rb −/− mice does not result from the absence of dermal CD11blow/−Langerin+CD103+ DCs.

Defining antigen-dependent stages of T cell migration from the blood to the central nervous system parenchyma

In experimental autoimmune encephalomyelitis (EAE), intravenous transfer of activated CD4+ myelin‐specific T cells is sufficient to induce disease. Transferred T cells access the CNS parenchyma by trafficking across the blood brain barrier (BBB) vascular endothelium into the perivascular space, and then across the glial limitans that is made up of astrocytes and microglia. Flow cytometry analysis of cells isolated from CNS tissue does not distinguish between T cell populations at the various stages of migration. In this study, we have used GK1.5 (anti‐CD4) treatment along with immunohistochemistry to distinguish between populations of T cells that are associated with the vasculature, T cells that have migrated into the perivascular space, and T cells in the parenchyma. We have also re‐evaluated antigen specificity requirements of T cells as they are recruited to the CNS parenchyma. Activated myelin‐specific T cells are restricted to the CNS vasculature for at least 24 h post transfer. MHC class II expression on the recipient is required for cells to traffic across the CNS vascular endothelium. Further, Con A‐stimulated or non‐CNS‐specific (ovalbumin‐specific) T cells fail to migrate into the perivascular space, and only enter the CNS parenchyma when co‐transferred with myelin‐specific T cells. Our results indicate that Th1 populations cannot accumulate in the perivascular (subarachnoid, Virchow‐Robbins) space without a CNS antigen‐specific signal.

Targeted knock-in mice expressing mutations of CD28 reveal an essential pathway for costimulation.

ABSTRACT Despite extensive study, the role of phosphatidylinositol 3-kinase (PI3-kinase) activation in CD28 function has been highly contentious. To definitively address this question, we generated knock-in mice expressing mutations in two critical domains of the cytoplasmic tail of CD28. Mutation of the proximal tyrosine motif interrupted PI3-kinase binding and prevented CD28-dependent phosphorylation of protein kinase B (PKB)/Akt; however, there was no detectable effect on interleukin-2 (IL-2) secretion, expression of Bcl-XL, or on T-cell function in vivo. Furthermore, we demonstrate that signaling initiated by the C-terminal proline motif is directly responsible for tyrosine phosphorylation of phosphoinosotide-dependent kinase 1, protein kinase Cθ, and glycogen synthase kinase 3β, as well as contributing to threonine phosphorylation of PKB. T cells mutated in this domain were profoundly impaired in IL-2 secretion, and the mice had marked impairment of humoral responses as well as less severe disease manifestations in experimental allergic encephalomyelitis. These data demonstrate that the distal proline motif initiates a critical nonredundant signaling pathway, whereas direct activation of PI3-kinase by the proximal tyrosine motif of CD28 is not required for normal T-cell function.

Bhlhe40 controls cytokine production by T cells and is essential for pathogenicity in autoimmune neuroinflammation

TH1 and TH17 cells mediate neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Pathogenic TH cells in EAE must produce the pro-inflammatory cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). TH cell pathogenicity in EAE is also regulated by cell-intrinsic production of the immunosuppressive cytokine interleukin 10 (IL-10). Here, we demonstrate that mice deficient for the basic helix-loop-helix (bHLH) transcription factor Bhlhe40 (Bhlhe40−/−) are resistant to the induction of EAE. Bhlhe40 is required in vivo in a T cell-intrinsic manner, where it positively regulates the production of GM-CSF and negatively regulates the production of IL-10. In vitro, GM-CSF secretion is selectively abrogated in polarized Bhlhe40−/− TH1 and TH17 cells, and these cells show increased production of IL-10. Blockade of IL-10 receptor in Bhlhe40−/− mice renders them susceptible to EAE. These findings identify Bhlhe40 as a critical regulator of autoreactive T cell pathogenicity.

B Cell Antigen Presentation Is Sufficient To Drive Neuroinflammation in an Animal Model of Multiple Sclerosis

B cells are increasingly regarded as integral to the pathogenesis of multiple sclerosis, in part as a result of the success of B cell–depletion therapy. Multiple B cell–dependent mechanisms contributing to inflammatory demyelination of the CNS have been explored using experimental autoimmune encephalomyelitis (EAE), a CD4 T cell–dependent animal model for multiple sclerosis. Although B cell Ag presentation was suggested to regulate CNS inflammation during EAE, direct evidence that B cells can independently support Ag-specific autoimmune responses by CD4 T cells in EAE is lacking. Using a newly developed murine model of in vivo conditional expression of MHC class II, we reported previously that encephalitogenic CD4 T cells are incapable of inducing EAE when B cells are the sole APC. In this study, we find that B cells cooperate with dendritic cells to enhance EAE severity resulting from myelin oligodendrocyte glycoprotein (MOG) immunization. Further, increasing the precursor frequency of MOG-specific B cells, but not the addition of soluble MOG-specific Ab, is sufficient to drive EAE in mice expressing MHCII by B cells alone. These data support a model in which expansion of Ag-specific B cells during CNS autoimmunity amplifies cognate interactions between B and CD4 T cells and have the capacity to independently drive neuroinflammation at later stages of disease.

Region-specific regulation of inflammation and pathogenesis in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis and is characterized by an infiltrate of predominantly T cells and macrophages in the spinal cord and brain. In both the spinal cord and the cerebellum, Th1 cells direct inflammation to antigen-rich white matter tracts, and there is a TNFR1-dependent recruitment of CD11b(hi) cells in both regions. In the spinal cord, parenchymal invasion, demyelination and clinical symptoms are associated with TNFR1-dependant parenchymal induction (especially astrocytes) of VCAM-1 and CXCL2. None of these events occur in the cerebellum despite the fact that an inflammatory infiltrate accumulates in the perivascular space. Therefore regional specificity in astrocyte responses to inflammatory cytokines may regulate regional parenchymal infiltration and pathogenesis.

Cutting Edge: Conditional MHC Class II Expression Reveals a Limited Role for B Cell Antigen Presentation in Primary and Secondary CD4 T Cell Responses

The activation, differentiation, and subsequent effector functions of CD4 T cells depend on interactions with a multitude of MHC class II (MHCII)–expressing APCs. To evaluate the individual contribution of various APCs to CD4 T cell function, we have designed a new murine tool for selective in vivo expression of MHCII in subsets of APCs. Conditional expression of MHCII in B cells was achieved using a cre-loxP approach. After i.v. or s.c. priming, partial proliferation and activation of CD4 T cells was observed in mice expressing MHCII only by B cells. Restricting MHCII expression to B cells constrained secondary CD4 T cell responses in vivo, as demonstrated in a CD4 T cell–dependent model of autoimmunity, experimental autoimmune encephalomyelitis. These results highlight the limitations of B cell Ag presentation during initiation and propagation of CD4 T cell function in vivo using a novel system to study individual APCs by the conditional expression of MHCII.