T Cell Interactions in Mycobacterial Granulomas: Non-Specific T Cells Regulate Mycobacteria-Specific T Cells in Granulomatous Lesions.

Infections with pathogenic mycobacteria are controlled by the formation of a unique structure known as a granuloma. The granuloma represents a host-pathogen interface where bacteria are killed and confined by the host response, but also where bacteria persist. Previous work has demonstrated that the T cell repertoire is heterogenous even at the single granuloma level. However, further work using pigeon cytochrome C (PCC) epitope-tagged BCG (PCC-BCG) and PCC-specific 5CC7 RAG-/- TCR transgenic (Tg) mice has demonstrated that a monoclonal T cell population is able to control infection. At the chronic stage of infection, granuloma-infiltrating T cells remain highly activated in wild-type mice, while T cells in the monoclonal T cell mice are anergic. We hypothesized that addition of an acutely activated non-specific T cell to the monoclonal T cell system could recapitulate the wild-type phenotype. Here we report that activated non-specific T cells have access to the granuloma and deliver a set of cytokines and chemokines to the lesions. Strikingly, non-specific T cells rescue BCG-specific T cells from anergy and enhance the function of BCG-specific T cells in the granuloma in the chronic phase of infection when bacterial antigen load is low. In addition, we find that these same non-specific T cells have an inhibitory effect on systemic BCG-specific T cells. Taken together, these data suggest that T cells non-specific for granuloma-inducing agents can alter the function of granuloma-specific T cells and have important roles in mycobacterial immunity and other granulomatous disorders.

Lung gene expression and single cell analyses reveal two subsets of idiopathic pulmonary fibrosis (IPF) patients associated with different pathogenic mechanisms.

Idiopathic pulmonary fibrosis is a progressive and debilitating lung disease with large unmet medical need and few treatment options. We describe an analysis connecting single cell gene expression with bulk gene expression-based subsetting of patient cohorts to identify IPF patient subsets with different underlying pathogenesis and cellular changes. We reproduced earlier findings indicating the existence of two major subsets in IPF and showed that these subsets display different alterations in cellular composition of the lung. We developed classifiers based on the cellular changes in disease to distinguish subsets. Specifically, we showed that one subset of IPF patients had significant increases in gene signature scores for myeloid cells versus a second subset that had significantly increased gene signature scores for ciliated epithelial cells, suggesting a differential pathogenesis among IPF subsets. Ligand-receptor analyses suggested there was a monocyte-macrophage chemoattractant axis (including potentially CCL2-CCR2 and CCL17-CCR4) among the myeloid-enriched IPF subset and a ciliated epithelium-derived chemokine axis (e.g. CCL15) among the ciliated epithelium-enriched IPF subset. We also found that these IPF subsets had differential expression of pirfenidone-responsive genes suggesting that our findings may provide an approach to identify patients with differential responses to pirfenidone and other drugs. We believe this work is an important step towards targeted therapies and biomarkers of response.

Mechanisms and regulation of IL-22-mediated intestinal epithelial homeostasis and repair.

AIMS: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. MAIN METHODS: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. KEY FINDINGS: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. SIGNIFICANCE: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.

Treatment with a CD40 Antagonist Antibody Reverses Severe Proteinuria and Loss of Saliva Production and Restores Glomerular Morphology in Murine Systemic Lupus Erythematosus.

CD40 is a costimulatory receptor on APCs that is critical for the induction and maintenance of humoral and cell-mediated immunity. Accordingly, CD40 and its ligand, CD40L, have long been considered targets for the treatment of autoimmune diseases. We developed a rat/mouse chimeric anti-mouse CD40 antagonist mAb, 201A3, and evaluated its ability to alleviate murine lupus. Treatment of NZB/W-F1 mice with 201A3 after the onset of severe proteinuria rapidly reversed established severe proteinuria and nephritis and largely restored normal glomerular and tubular morphology. This coincided with a normalization of the expression of genes associated with proteinuria and injury by kidney parenchymal cells. Anti-CD40 treatment also prevented and reversed loss of saliva production and sialadenitis. These effects on kidney and salivary gland function were confirmed using mice of a second strain, MRL/Mp-lpr/lpr, and extended to alleviating joint inflammation. Immunologically, anti-CD40 treatment disrupted multiple processes that contribute to the pathogenesis of systemic lupus erythematosus (SLE), including autoreactive B cell activation, T effector cell function in target tissues, and type I IFN production. This ability to disrupt disease-critical immunological mechanisms, to reverse glomerular and tubular injury at the cellular and gene expression levels, and to confer exceptional therapeutic efficacy suggests that CD40 is a central disease pathway in murine SLE. Thus, a CD40 antagonist Ab could be an effective therapeutic in the treatment of SLE.

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.

Bat3 promotes T cell responses and autoimmunity by repressing Tim-3­mediated cell death and exhaustion.

T cell immunoglobulin and mucin domain­containing 3 (Tim-3) is an inhibitory receptor that is expressed on exhausted T cells during infection with HIV-1 and hepatitis C virus. By contrast, Tim-3 expression and function are defective in multiple human autoimmune diseases. However, the molecular mechanisms modulating Tim-3 function are not well understood. Here we show that human leukocyte antigen B (HLA-B)-associated transcript 3 (Bat3) binds to, and represses the function of, Tim-3. Bat3 protects T helper type 1 (TH1) cells from galectin-9­mediated cell death and promotes both proliferation and proinflammatory cytokine production. Bat3-deficient T cells have elevated expression of exhaustion-associated molecules such as Tim-3, Lag3, Prdm1 and Pbx3, and Bat3 knockdown in myelin-antigen­specific CD4+ T cells markedly inhibits the development of experimental autoimmune encephalomyelitis while promoting the expansion of a dysfunctional Tim-3hi, interferon-γ (IFN-γ)loCD4+ cell population. Furthermore, expression of Bat3 is reduced in exhausted Tim-3+ T cells from mouse tumors and HIV-1­infected individuals. These data indicate that Bat3 acts as an inhibitor of Tim-3­dependent exhaustion and cell death. Bat3 may thus represent a viable therapeutic target in autoimmune disorders, chronic infections and cancers.

Proteasome inhibition is partially effective in attenuating pre-existing immunity against recombinant adeno-associated viral vectors.

Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezomib efficiently reduces AAV-specific IgG titres and moderates the cytotoxic T cell response in mice that have a pre-existing immunity to AAV2/8. Significant depletion of AAV2/8-specific IgG-producing plasma cells in secondary lymphoid organs and bone marrow was observed. However, this inhibition of the immune response by bortezomib was insufficient to allow subsequent re-infection with a recombinant AAV vector of a similar serotype. We show that this shortcoming is probably due to the combination of residual antibody levels and the inability of bortezomib to completely deplete the memory B cells that are re-activated in response to a repeated infection with a recombinant AAV vector. Taken together, the results of this study argue for the use of immunosuppressive therapies that target both plasma and memory B cells for the efficient elimination of pre-existing immunity against AAV2/8 vectors.

Ligation of cytotoxic T lymphocyte antigen-4 to T cell receptor inhibits T cell activation and directs differentiation into Foxp3+ regulatory T cells.

Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-ß and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-ß. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.

Tim-3/galectin-9 pathway: regulation of Th1 immunity through promotion of CD11b+Ly-6G+ myeloid cells.

IFN-gamma plays a central role in antitumor immunity. T cell Ig and mucin domain (Tim-3) is expressed on IFN-gamma-producing Th1 cells; on interaction with its ligand, galectin-9, Th1 immunity is terminated. In this study, we show that transgenic overexpression of Tim-3 on T cells results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Molecular characterization of CD11b(+)Ly-6G(+) cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells. Accordingly, we find that modulation of the Tim-3/galectin-9 (Gal-9) pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, Gal-9, results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b(+)Ly-6G(+) cells and normal immune responses in Gal-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where myeloid-derived suppressor cells are disadvantageous.

Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells.

CD4+ T helper 1 (TH1) cells are important mediators of inflammation and are regulated by numerous pathways, including the negative immune receptor Tim-3. We found that Tim-3 is constitutively expressed on cells of the innate immune system in both mice and humans, and that it can synergize with Toll-like receptors. Moreover, an antibody agonist of Tim-3 acted as an adjuvant during induced immune responses, and Tim-3 ligation induced distinct signaling events in T cells and dendritic cells; the latter finding could explain the apparent divergent functions of Tim-3 in these cell types. Thus, by virtue of differential expression on innate versus adaptive immune cells, Tim-3 can either promote or terminate TH1 immunity and may be able to influence a range of inflammatory conditions.

Virally activated CD8 T cells home to Mycobacterium bovis BCG-induced granulomas but enhance antimycobacterial protection only in immunodeficient mice.

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-gamma)-producing LCMV-specific T cells in liver granulomas and increased local IFN-gamma. 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.

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-gamma secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-gamma 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.

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-gamma and IL-17. The main source of local TNF-alpha 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.

Immediate DNA ploidy analysis of gastrointestinal biopsies taken by endoscopy using a mechanical dissociation device.

BACKGROUND: Quantitative DNA analysis of fresh biopsy material can contribute to a more accurate diagnosis and prognosis. The authors aimed to develop and test a mechanical, nuclear preparation protocol for quantitative DNA analysis. PATIENTS AND METHODS: Altogether 32 gastric (10 healthy, 17 gastritis, 7 adenocarcinoma) and 48 colon (21 healthy, 20 colitis ulcerosa, 7 adenocarcinoma) biopsy specimens were evaluated. The mechanical disruption was performed by Medimachine (DAKO, Denmark). The flow cytometry analysis was performed on a BD FACSStar flow cytometer. RESULTS: DNA Aneuploidy was found in gastric samples only in tumours. The S-phase fraction of the normal cases was 5.9 +/- 2.1%, 5.1% +/- 1.2% in gastritis and 10.7 +/- 1.6% in carcinomas. Seven out of 20 colitis ulcerosa and 4 out of 7 colon cancer samples were aneuploid. The S-phase fraction of normal colon cases was 5.7 +/- 3.4%, in colitis 8.1 +/- 4.2% and 15.1 +/- 5.7% in carcinomas, respectively. CONCLUSION: Mechanical nuclear isolation is a useful method for flow cytometric DNA ploidy analysis of fresh biopsy samples.