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DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling in immune-mediated inflammatory diseases (IMIDs), including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC) by analyzing the expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control ( n = 31), inactive UC ( n = 31), active UC ( n = 33), and a UC single-cell RNA-Seq dataset. The effects of type I IFN (IFN-β), IFN-γ, and TNF-α on gene expression, cytokine production, and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1β/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 ( AIM2 ), IFI16 , myeloid cell nuclear differentiation antigen ( MNDA ), and pyrin and HIN domain family member 1 ( PYHIN1 )- as well as Z-DNA-binding protein 1 ( ZBP1 ), cyclic GMP-AMP synthase ( cGAS ), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes ( CASP1 , IL1B , and IL18 ), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 ( STING ), TBK1 , and IRF3 ), IFNB1 , and type I IFN biomarker genes ( OAS2 , IFIT2 , and MX2 ) were also increased in active UC. Cotreatment of organoids with IFN-β or IFN-γ in combination with TNFα increased expression of IFI16 , ZBP1 , CASP1 , cGAS , and STING induced cell death and IL-1β/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC. NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-β, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.

Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal epithelial barrier leading to breach of barrier immunity. Here we identified similar protein expression changes between IBD and Citrobacter rodentium-infected FVB mice with respect to dysregulation of solute transporters as well as components critical for intestinal barrier integrity. We attribute the disease associated changes in the model to the emergence of undifferentiated intermediate intestinal epithelial cells. Prophylactic treatment with IL-22.Fc in C. rodentium-infected FVB mice reduced disease severity and rescued the mice from lethality. Multi-omics and solute analyses revealed that IL-22.Fc treatment prevented disease-associated changes including disruption of the solute transporter machinery and restored proper physiological functions of the intestine, respectively. Taken together, we established the disease relevance of the C. rodentium-induced colitis model to IBD, demonstrated the protective role of IL-22 in amelioration of epithelial dysfunction and elucidated the molecular mechanisms with IL-22's effect on intestinal epithelial cells., (© 2022. The Author(s), under exclusive licence to Society for Mucosal Immunology.)

Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn's disease (CD). Th1-type cytokines-IFN-γ and TNF-α-occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs., (© 2021. The Author(s).)

Intestinal permeability and neutrophil activity are closely linked to inflammatory bowel disease (IBD) pathophysiology. Here we discuss two techniques for assessing permeability and neutrophil activity in mouse IBD models using near infrared (NIR) detection. To address the limitation of visible light readouts-namely high background-IRDye 800CW was used to enable rapid, non-terminal measurements of intestinal permeability. The increased sensitivity of NIR readouts for colon permeability is shown using dextran sulfate sodium (DSS) and anti-CD40 murine colitis models in response to interleukin-22 immunoglobulin Fc (IL22Fc) fusion protein and anti-p40 monoclonal antibody treatments, respectively. In addition to enhanced permeability, elevated levels of neutrophil elastase (NE) have been reported in inflamed colonic mucosal tissue. Activatable NIR fluorescent probes have been extensively used for disease activity evaluation in oncologic animal models, and we demonstrate their translatability using a NE-activatable reagent to evaluate inflammation in DSS mice. Confocal laser endomicroscopy (CLE) and tissue imaging allow visualization of spatial NE activity throughout diseased colon as well as changes in disease severity from IL22Fc treatment. Our findings with the 800CW dye and the NE probe highlight the ease of their implementation in preclinical IBD research.

Background: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications., Results: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies., Conclusions: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.

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-F 1 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., (Copyright © 2019 by The American Association of Immunologists, Inc.)

In inflammatory bowel disease (IBD), inflammation is sustained by an exaggerated response of lymphocytes. This results from enhanced expression of anti-apoptotic B cell lymphoma (BCL-2) and BCL-XL associated with a diminished turnover. Azathioprine (AZA) directly targets BCL-2 family-mediated apoptosis. We investigated whether the BCL-2 family expression pattern could be used to predict treatment response to AZA and determined whether BCL-2 inhibitor A-1211212 effectively diminishes lymphocytes and ameliorates inflammation in a model of colitis. BCL-2 family expression pattern was determined by next-generation sequencing (NGS). BCL-2 inhibitor was administered orally to Il10-/- mice. Haematological analyses were performed with an ADVIA 2120 and changes in immune cells were investigated using quantitative polymerase chain reaction (qPCR) and fluorescence activated cell sorter (FACS). We determined similar expression levels of BCL-2 family members in patients with remission and patients refractory to treatment, showing that BCL-2 family expression can not predict AZA treatment response. Expression was not correlated with the modified Truelove and Witts activity index (MTWAI). BCL-2 inhibitor initiated cell death in T cells from patients refractory to AZA and reduced lymphocyte count in Il10-/- mice. FACS revealed diminished CD8 + T cells upon BCL-2 inhibitor in Il10-/- mice without influencing platelets. Tnf, Il1β, IfnƔ and Mcp-1 were decreased upon BCL-2 inhibitor. A-1211212 positively altered the colonic mucosa and ameliorated inflammation in mice. Pro-apoptotic BCL-2 inhibitor A-1211212 diminishes lymphocytes and ameliorates colitis in Il10-/- mice without inducing thrombocytopenia. BCL-2 inhibition could be a new therapy option for patients refractory to AZA., (© 2018 British Society for Immunology.)

Background & Aims: Although tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis., Methods: We generated Rag1 -/- mice that lack all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR -/- Rag1 -/- mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RB hi to FcγR -/- Rag1 -/- or Rag1 -/- littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206 + (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay., Results: Rag1 -/- mice with colitis given anti-TNF had near complete mucosal healing and Rag1 -/- mice given an isotype control antibody developed severe colitis. In contrast, FcγR -/- Rag1 -/- mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR -/- Rag1 -/- mice given a control antibody. Colons from Rag1 -/- mice that received anti-TNF had an increased number of CD206 + macrophages compared with Rag1 -/- mice given control antibody; in FcγR -/- Rag1 -/- mice given anti-TNF these numbers were as low as FcγR -/- Rag1 -/- given the control antibody. In human PBMCs, anti-TNF increased the number of CD206 + macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206 + macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206 + macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206 + macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology., Conclusions: In a study of the in vitro and in vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206 + macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206 + macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Anti-tumour necrosis factor [TNF] monoclonal antibodies [infliximab, adalimumab] induce complete mucosal healing in a proportion of patients with Crohn's disease whereas a TNF receptor fusion protein [etanercept] is not effective and the anti-TNF F[ab']2 fragment [certolizumab] shows a very low rate of complete mucosal healing. In contrast, all four TNF-neutralising drugs have demonstrated efficacy in the treatment of rheumatoid arthritis. These observations suggest that factors other than neutralisation of TNF may contribute to clinical outcomes in Crohn's disease. Here we tested the hypothesis that Fc receptor [FcR]-mediated effects may contribute to the therapeutic response of anti-TNF antibodies in inflammatory bowel disease., Methods: We modified an IgG2c mouse anti-TNF antibody that binds the high-affinity FcRs to generate an IgG1 isotype with strongly diminished binding. We examined the therapeutic effects of both antibodies in the T cell transfer model of inflammatory bowel disease and the collagen-induced arthritis model., Results: The IgG2c anti-TNF antibody prevented colonic inflammation in the T cell transfer model of colitis, whereas the IgG1 anti-TNF did not. Conversely, both the IgG2c and IgG1 anti-TNFs were similarly effective in reducing the severity of articular inflammation in mouse collagen-induced arthritis., Conclusion: These data support the concept that the mechanism of action for TNF-neutralising drugs may differ across immune-mediated diseases and, potentially, between therapeutics within a particular disease. Our data suggest a specific role of Fc-mediated immune regulation in the resolution of intestinal inflammation by anti-TNF monoclonal antibodies., (Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Exaggerated activation of lymphocytes contributes to the pathogenesis of inflammatory bowel disease (IBD). Medical therapies are linked to the BCL-2 family-mediated apoptosis. Imbalance in BCL-2 family proteins may cause failure in therapeutic responses. We investigated the role of BCL-2 inhibitor ABT-737 for lymphocyte apoptosis in mice under inflammatory conditions. B.6129P2-interleukin (IL)-10(tm1Cgn) /J (IL-10(-/-) ) weighing 25-30 g with ongoing colitis were used. Fifty mg/kg/day ABT-737 was injected intraperitoneally (i.p.). Haematological analyses were performed with an ADVIA 2120 flow cytometer and mass cytometry with a CyTOF 2. Following i.p. administration, ABT-737 was detected in both spontaneous and acute colitis in peripheral blood (PBL) and colon tissue. Treatment led to lymphopenia. CD4(+) CD44(+) CD62L(+) central memory and CD8(+) , CD44(+) CD62L(-) central memory T cells were decreased in PBL upon ABT-737 compared to vehicle-receiving controls. Increased apoptosis upon ABT-737 was determined in blood lymphocytes, splenocytes and Peyer's patches and was accompanied by a decrease in TNF and IL-1B. ABT-737 positively altered the colonic mucosa and ameliorated inflammation, as shown by colonoscopy, histology and colon length. A decreased BIM/BCL-2 ratio or absence of BIM in both Bim(-) (/) (-) and Il10(-) (/) (-) × Bim(-) (/) (-) impeded the protective effect of ABT-737. The BIM/BCL-2 ratio decreased with age and during the course of treatment. Thus, long-term treatment resulted in adapted TNF levels and macroscopic mucosal damage. ABT-737 was efficacious in diminishing lymphocytes and ameliorating colitis in a BIM-dependent manner. Regulation of inappropriate survival of lymphocytes by ABT-737 may provide a therapeutic strategy in IBD., (© 2015 British Society for Immunology.)

In multiple sclerosis, long-term disability is caused by axonal and neuronal damage. Established therapies target primarily the inflammatory component of the disease, but fail to prevent neurodegeneration. Fingolimod (codenamed FTY720) is an oral sphingosine 1-phosphate (S1P) receptor modulator with promising results in phase II trials in multiple sclerosis patients and is under further development as a novel treatment for multiple sclerosis. To evaluate whether FTY720 has neuroprotective properties, we tested this drug in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. FTY720 exerted significant anti-inflammatory effects during optic neuritis and reduced inflammation, demyelination, and axonal damage; however, FTY720 treatment did not prevent apoptosis of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. Consistent with this lack of effect on RGC survival, FTY720 treatment did not improve visual function, nor did it prevent apoptosis of RGCs in vitro. We observed a persistent activation of apoptotic signaling pathways in RGCs under FTY720 treatment, a possible underlying mechanism for the lack of neuroprotection in the presence of strong anti-inflammatory effects, Furthermore, FTY720 shifted the remaining inflammation in the optic nerve toward neurotoxicity by modest up-regulation of potential neurotoxic cytokines. We conclude that FTY720-induced anti-inflammation and axon protection did not of itself protect neurons from apoptotic cell death., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

USPIO-enhanced MRI allows non-invasive visualization of mononuclear cell infiltration into CNS lesions in MS and EAE. Herein, we show a distinct spatiotemporal pattern of CNS lesions that reveals the involvement of spino-olivocerebellar pathways in MOG-induced EAE rats using USPIO-enhanced MRI. Specifically, lesions of the inferior olives were observed primarily in the acute phase whereas lesions of cerebellum or spinal cord/brainstem were observed during the relapse phase. Further, behavioral deficits observed from these animals are consistent with the functional role of spino-olivocerebellar pathways in coordination and movement. Collectively, our results provide new insights into the pathophysiology of this animal model of MS.

The Src family kinases Lck and Fyn play an important role in T cell development and function. We have synthesized a novel small molecule, A-420983, which inhibits Lck and Fyn, as well as other Src family kinases, but has selectivity with respect to non-Src family kinases. A-420983 completely inhibited antigen-stimulated production of IFN-gamma and IL-4 by mouse Th1 and Th2 cells, respectively. Antigen-induced T cell proliferation was also blocked by treatment with A-420983. In contrast, IL-15-induced proliferation was unaffected by A-420983, suggesting that TCR-independent pathways of T cell activation were not impaired. When mice were dosed orally, A-420983 inhibited TCR-mediated c-jun and ZAP-70 phosphorylation in CD4+ T cells and suppressed the disease course of established EAE. Treatment with A-420983 for 7 days resulted in a block in thymocyte development at the CD4- CD8- stage, consistent with inhibition of Lck and Fyn in vivo. These results demonstrate that a small molecule inhibitor of Lck and Fyn can block TCR-induced T cell activation in vitro and in vivo. Furthermore, CNS demyelination mediated by activated encephalitogenic CD4+ T cells is dependent upon the kinase activity of these Src family members. We conclude that inhibition of Src family kinases may represent a promising strategy for the treatment of T cell-mediated disorders.

Dendritic cell (DC) precursors and immature DC reside in epithelium where they encounter pathogens and cytokines, which stimulate their differentiation. We hypothesized that type-I interferons (IFN-alpha and -beta), cytokines that are produced early in the innate immune response against viruses and some bacteria, may influence DC differentiation and function. To examine this possibility, we used an in vitro model of DC differentiation in which initial culture of human CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 generates immature DC, and subsequent culture with tumor necrosis factor (TNF)-alpha drives the final development into mature DC. We found in this model that IFN-alpha/beta, added from the initiation of the culture on, significantly reduced the survival and altered the morphology and differentiation of DC. TNF-alpha-dependent maturation of IFN-beta-treated immature DC led to cells with reduced expression of CD1a, CD40, CD54, and CD80 when compared with mature DC controls. IFN-alpha/beta-treated DC further had a reduced capacity to induce naive Th-cell proliferation through allostimulation or anti-CD3 monoclonal antibody stimulation. In addition, IFN-alpha/beta-treated DC secreted less IL-12 upon stimulation with Staphylococcus aureus Cowan strain or with CD4(+) T cells, and this decrease correlated directly with their inability to support CD4(+) T-cell secretion of IFN-gamma, even though T-cell lymphotoxin production was unaffected. These findings indicate that type-I IFNs can influence the generation of acquired immune responses by modifying T-helper cell differentiation through the regulation of DC differentiation and function.

We have previously shown that IFN-beta, a key cytokine associated with the early phase of the innate host defense, can prevent the generation of human Th1 cells. Specifically, we demonstrated that IFN-beta prevents the in vitro monocyte-derived mature dendritic cell (DC)-dependent differentiation of naive Th cells into IFN-gamma-secreting Th cells, as a result of its ability to inhibit DC IL-12 secretion. The goal of the present study was to identify how IFN-beta negatively regulates IL-12 secretion by DC. We report that in our Th cell differentiation model, DC IL-12 secretion is dependent on the CD40L/CD40 accessory pathway, and, utilizing a Th cell-free system, we find that IFN-beta inhibits anti-CD40 mAb-induced DC secretion of the p40 chain of the IL-12 heterodimer. In addition, we show that IFN-beta-mediated inhibition of CD40 signaling does not interfere with all signaling pathways emanating from CD40, since anti-CD40 mAb-induced DC IL-6 secretion is augmented by IFN-beta. Thus, our results demonstrate that signaling from CD40 is differentially regulated by IFN-beta. A second critical element of innate immunity involves the response against components of bacterial membranes such as LPS. DC respond to LPS by secreting IL-6 and IL-12. In contrast to CD40-dependent IL-6 and IL-12 secretion, we find that LPS-induced DC secretion of p40 IL-12 and IL-6 is not affected by IFN-beta. Our findings show that IFN-beta influences the generation of acquired immune responses through its regulation of CD40-dependent DC functions.

Mycosis fungoides is a low-grade cutaneous T-cell lymphoma. Early treatment often involves the use of topical chemotherapy such as mechlorethamine or carmustine although single-agent oral chemotherapy with alkylators is common for advanced disease. Recently, in a Phase I study of the new alkylating agent temozolomide, two mycosis fungoides patients experienced a complete response. The mechanism of resistance to alkylating drugs such as temozolomide is thought to be due to the presence in tumor cells of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT). The protein mediates a reaction with the O6-position of guanine in DNA, removing the lesion and leaving guanine intact. We, therefore, examined the levels of AGT in CD4+ T lymphocytes obtained by negative antibody selection from the blood of noncancerous individuals and mycosis fungoides patients, and in paraffin-embedded sections from mycosis fungoides patch, plaque, or tumor lesions and cells from involved lymph nodes. AGT protein levels were measured by quantitative immunofluorescence microscopy using a monoclonal antibody against human AGT. Using this approach, the mean level of our positive control (AGT-expressing cells) was 84,807 molecules/nucleus; values below 5,000 molecules/nucleus are considered very low. The mean AGT level in CD4+ T lymphocytes from noncancerous and cancerous individuals was 18,618 (n = 12) and 8,593 (n = 5), respectively. The mean fraction of outliers in circulating CD4+ T lymphocytes from mycosis fungoides patients was statistically significantly lower than T cells in lymph nodes. AGT molecules/nucleus from lymph node biopsies from 8 of 10 patients showed low (< 10,000 molecules/nucleus) or undetectable levels (n = 5) of AGT. The mean AGT level from samples of mycosis fungoides patch/plaque and tumor was also low at 221 (n = 4) and 2,363 (n = 6), respectively. Surprisingly, Hut78, a mycosis fungoides T-cell lymphoma cell line, was positive for AGT activity (median: 77,700 molecules/nucleus), and Hut102--another mycosis fungoides cell line--was low (median: 5,990 molecules/nucleus). Because AGT is a primary means of cell resistance to alkylating agents, the low level of AGT in neoplastic T lymphocytes from patients with mycosis fungoides suggests that treatment with alkylating agents producing O6-alkylguanine adducts, such as carmustine or temozolomide, may produce improved clinical outcomes.

We have investigated the role of type I IFNs (IFN-alpha and -beta) in human T cell differentiation using anti-CD3 mAb and allogeneic, in vitro-derived dendritic cells (DC) as APCs. DC were very efficient activators of naive CD4+ T cells, providing necessary costimulation and soluble factors to support Th1 differentiation and expansion. Addition of IFN-alphabeta to DC/T cell cultures resulted in induction of T cell IL-10 production and inhibition of IFN-gamma, TNF-alpha, and LT secretion. Diminished T cell IFN-gamma production correlated with IFN-alphabeta-mediated inhibition of the p40 chain of the IL-12 heterodimer secreted by DC. Suppression of p40 IL-12 and IFN-gamma was not due to increased levels of IL-10 in these cultures, and production of IFN-gamma could be restored by exogenous IL-12. These data indicate that type I IFNs inhibit DC p40 IL-12 expression, which is required for development of IFN-gamma-producing CD4+ T cells. Furthermore, when T cells were restimulated without IFN-beta, these cells induced less p40 IL-12 from DC, suggesting that the functional properties of T cells may regulate DC function. Thus, IFN-alphabeta inhibits both IL-12-dependent and independent Th1 cytokine production and provides a mechanism for inhibition of IL-12-mediated immunity in viral infections.

Our previous work has shown that specific peripheral immune tolerance induced by the intravenous administration of ECDI-fixed, antigen-coupled syngeneic splenocytes is an extremely efficient method for prevention and treatment of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE) in susceptible SJL/J mice. The current study examined the mechanisms by which unresponsiveness is induced in primed encephalitogenic T cells. The results indicate that the inhibition of MBP-specific T cells by the i.v. injection of MBP-coupled splenocytes is not due to the induction of antigen-specific regulatory T cells, but rather to the induction of anergy/deletion of the effector cells. This conclusion is supported by the findings that spleen or lymph node cells isolated from MBP-tolerant mice fail to inhibit the adoptive transfer of R-EAE in cotransfer assays, and that tolerance is not inhibited by prior thymectomy or prior treatment with cyclophosphamide or anti-CD8 monoclonal antibody. In contrast, we demonstrate that splenocytes from MBP-tolerized, asymptomatic mice have a significantly reduced ability to serially transfer R-EAE to naive secondary recipients following antigen re-activation in vitro, in the first several weeks following tolerization, but that the ability to serially transfer R-EAE returns to sham tolerant control levels within 1-2 months. We also demonstrate a significantly reduced precursor frequency of MBP-specific, IL-2-producing T cells in the MBP-tolerant within three days of treatment. Collectively, the data most closely support a model wherein inhibition of MBP-specific encephalitogenic CD4+ effector T cells by i.v. injected MBP-coupled splenocytes is due to the direct induction of anergy/deletion from which they can recover over time.

Type I interferons (IFN) are important regulators of both innate and acquired immunity. We have used an in vitro system of human CD4+ T cell differentiation to determine how IFN-beta influences development of T helper (Th) subsets and homing receptor expression. IFN-beta promoted differentiation of CD4+ T cells that produce low levels of both IFN-gamma and lymphotoxin compared to interleukin (IL)-12-derived Th1 CD4+ T cells. IFN-beta inhibited production of Th2 cytokines (IL-5 and IL-13) and augmented IL-12-mediated IL-10 secretion. In addition, IFN-beta significantly enhanced L-selection expression on CD4+ T cells and synergized with IL-12 to induce expression of cutaneous lymphocyte-associated antigen (CLA). This Th1 L-selectin+, CLA+ phenotype is characteristic of T cells found in normal human skin and suggests a role for type I IFN in the regulation of Th subset differentiation and tissue-specific homing receptors.

Peptide-specific tolerance with PLP139-151 peptide analogs was used to compare the fine antigen-specificity requirements at both the inductive and effector phases of relapsing EAE (R-EAE). A PLP139-151 analog peptide containing a single substitution at the primary T cell receptor (TcR) contact residue (A144) did not induce proliferation in PLP139-151-primed CD4+ T cells. In addition, tolerance induced with ECDI-treated. A144-coupled splenocytes failed to prevent the inductive phase of PLP139-151-induced R-EAE or to inhibit the induction of peptide-specific DTH indicating that naive PLP139-151-specific T cells do not react with the A144 peptide analog. In contrast, A144-coupled splenocytes did prevent the expression of the effector phase of R-EAE and inhibited the elicitation of peptide-specific DTH responses upon administration to mice seven days after immunization with PLP139-151. The results provide in vivo evidence that "antigen-experienced' T cells recognize a broader repertoire of antigens than do naive T cells and have important implications for the regulation of immune responses and for advancing our understanding of the pathogenesis and treatment of autoimmune disease.

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated, inflammatory demyelinating disease of the central nervous system (CNS) that serves as a model for the human demyelinating disease, multiple sclerosis. A critical event in the pathogenesis of EAE is the entry of both Ag-specific T lymphocytes and Ag-nonspecific mononuclear cells into the CNS. In the present report we investigated the role of two C-C chemokines (macrophage inflammatory protein-1 alpha (MIP-1 alpha) and monocyte chemotactic protein-1) and a C-x-C chemokine (MIP-2) in the pathogenesis of EAE. Production in the CNS of MIP-1 alpha, but not that of MIP-2, a rodent homologue of IL-8, or monocyte chemotactic protein-1, correlated with development of severe clinical disease. Administration of anti-MIP-1 alpha, but not that of anti-monocyte chemotactic protein-1, prevented the development of both acute and relapsing paralytic disease as well as infiltration of mononuclear cells into the CNS initiated by the transfer of neuroantigen peptide-activated T cells. Ab therapy could also be used to ameliorate the severity of ongoing clinical disease. Anti-MIP-1 alpha did not affect the activation of encepahlitogenic T cells as measured by cytokine secretion, surface marker expression, and ability to adoptively transfer EAE. These results demonstrate that MIP-1 alpha plays an important role in directing the chemoattraction of mononuclear inflammatory cells in the T cell-mediated autoimmune disease, EAE.

CD4+ T cells specific for PLP 139-151 induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease multiple sclerosis (MS) in both clinical course and histopathology. Conservative and nonconservative amino acid substitutions were introduced at three TcR or MHC contact residues within PLP 139-151 to identify fine specificity requirements, at the polyclonal level, for stimulating naive encephalitogenic T cells and for reactivating pre-primed autoreactive T cells as measured by T cell proliferation, cytokine induction, and functional encephalitogenic potential. The results indicate that peptides with substitutions at position 145 exhibited a significantly diminished ability to induce active disease, but these substitutions had little or no effect on the ability to activate PLP 139-151-primed T cells for proliferation or disease transfer. A conservative or a nonconservative substitution at position 144 ablated both encephalitogenic potential in active and adoptive EAE models and the ability to induce proliferative responses in T cells primed to the native peptide. A nonconservative lysine for glycine, but not a conservative serine substitution, at position 146 had similar effects. In contrast to their inability to induce active EAE and stimulate in vitro proliferation of PLP 139-151-primed T cells, the Y144 and the 146 analog peptides were able to suboptimally reactivate these cells for transfer of adoptive EAE. Furthermore, the nonencephalitogenic K146 peptide was found to exacerbate in vivo induction of EAE induced by priming with a suboptimal dose of PLP 139-151. These data support the hypothesis that naive neuroantigen-specific CD4+ T cells have more stringent activation requirements than do PLP 139-151-specific T cells which have previously encountered antigen. The finding that the analog peptides induced differential patterns of cytokine production, with LT/TNF-alpha production but not IFN-gamma production correlating with full encephalitogenic potential, suggests different functional outcomes may result from differential levels of signal transduction triggered by the substituted peptides. The significance of these results to the potential development of autoimmune disease via molecular mimicry and for the development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed.

The role of epitope spreading in the pathology of relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) was examined. Using peripherally induced immunologic tolerance as a probe to analyze the neuropathologic T cell repertoire, we show that the majority of the immunopathologic reactivity during the acute phase of R-EAE in SJL/J mice induced by active immunization with the intact proteolipid (PLP) molecule is directed at the PLP139-151 epitope and that responses to secondary encephalitogenic PLP epitopes may contribute to the later relapsing phases of disease. Intermolecular epitope spreading was demonstrated by showing the development of T cell responses to PLP139-151 after acute disease in mice in which R-EAE was initiated by the transfer of T cells specific for the non-cross-reactive MBP84-104 determinant. Intramolecular epitope spreading was demonstrated by showing that endogenous host T cells specific for a secondary encephalitogenic PLP epitope (PLP178-191) are demonstrable by both splenic T cell proliferative and in vivo delayed-type hypersensitivity responses in mice in which acute central nervous system damage was initiated by T cells reactive with the immunodominant, non-cross-reactive PLP139-151 sequence. The PLP178-191-specific responses are activated as a result of and correlate with the degree of acute tissue damage, since they do not develop in mice tolerized to the initiating epitope before expression of acute disease. Most importantly, we show that the PLP178-191-specific responses are capable of mediating R-EAE upon adoptive secondary transfer to naive recipient mice. Furthermore, induction of tolerance to intact PLP (which inhibits responses to both the initiating PLP139-151 epitope and to the PLP178-191 epitope) after the acute disease episode is sufficient to prevent relapsing disease. These results strongly support a contributory role of T cell responses to epitopes released as a result of acute tissue damage to the immunopathogenesis of relapsing clinical episodes and have important implications for the design of antigen-specific immunotherapies for the treatment of chronic autoimmune disorders in humans.

Fig. 6 depicts a model for epitope spreading in T cell-mediated demyelination. The acute phase of disease is due to T cells specific for the initiating epitope, which can be either a determinant on the CNS target organ of the autoimmune response or a determinant on a persisting, CNS-tropic virus. The primary T cell response is responsible for the initial tissue damage by the production of proinflammatory Th1 cytokines which can affect myelination directly (Selmaj et al. 1991) and indirectly by their ability to recruit and activate macrophages to phagocytize myelin (Cammer et al. 1978). As a result of myelin damage and opening of the blood-brain-barrier during acute disease, T cells specific for endogenous epitopes on the same and/or different myelin proteins are primed and expand either in the periphery or locally in the CNS. These secondary T cells initiate an additional round of myelin destruction, leading to a clinical relapse by production of additional pro-inflammatory cytokines, similar to the bystander demyelination operative during acute disease. It will be of great interest to determine the relative contributions of local and systemic immune responses to these endogenous neuroepitopes. It is possible that local CNS presentation of endogenous neuroepitopes following acute CNS damage could be mediated by infiltrating inflammatory macrophages, activated microglial cells, endothelial cells and/or astrocytes. These tissue resident antigen presenting cells have been shown to upregulate expression of MHC class II (Sakai et al. 1986, Traugott & Lebon 1988), certain adhesion molecules (Cannella et al. 1990), and B7 costimulatory molecules (K. M. Nikcevich, J. A. Bluestone, and S. D. Miller, in preparation) in response to pro-inflammatory cytokines. The data on epitope spreading provided by the murine demyelinating disease models clearly illustrate the dynamic nature of the T cell repertoire during chronic inflammation in a specific target organ. The contribution of epitope spreading to chronic CNS demyelination could be considered to be a special case since tolerance to myelin epitopes would be expected to be inefficient due to their sequestration behind the blood-brain-barrier. However, the recent description of epitope spreading in response to pancreatic antigens in spontaneous diabetes in the NOD mouse may indicate that this phenomenon is operative in a variety of organ-specific experimental and spontaneous autoimmune diseases.(ABSTRACT TRUNCATED AT 400 WORDS)

PLP 139-151(S) is the major encephalitogenic epitope of PLP in the SJL/J mouse. CD4+ T cells specific for PLP 139-151(S) induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease MS in both clinical course and histopathology. We are interested in events involved in activation of autoreactive T cells and how to specifically regulate these immune response to both prevent and treat ongoing demyelinating disease. In the current study, we examined the effect of both amino acid substitutions and deletions in the native PLP 139-151(S) peptide to identify which residues are critical for immunogenicity and encephalitogenicity. Conservative and nonconservative substitutions at position 145 diminished or completely destroyed the encephalitogenic potential of the peptide without affecting the ability to recall a proliferative response in lymph node T cells primed with the native PLP 139-151(S) peptide indicating an interesting dichotomy between ability to induce T cell proliferation and ability to induce active clinical disease. In addition, tryptophan at position 144 was identified as a critical TCR contact site as a peptide containing an alanine for tryptophan at this position (A144) primed a unique population of T cells which did not cross react with the native PLP 139-151(S). In addition, A144 was unable to stimulate PLP 139-151(S)-specific T cells in vitro or to induce active relapsing EAE in vivo. The significance of these results to the potential development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed.

Proteolipid protein (PLP) is a major component of the central nervous system (CNS) myelin membrane and has been shown to induce acute experimental autoimmune encephalomyelitis (EAE) in genetically susceptible animals. Here we describe conditions by which a relapsing-remitting form of EAE can be reliably induced in SJL/J mice either actively immunized with the major encephalitogenic PLP peptide, PLP13-151(S), or following adoptive transfer of PLP139-151(S)-specific T cells. The disease follows a reliable relapsing-remitting course with acute clinical signs first appearing 6-20 days after priming or transfer and relapses first appearing at 30-45 days. The initial onset of disease correlates with delayed-type hypersensitivity (DTH) reactivity specific for PLP139-151(S), in the apparent absence of T cell reactivity to the major myelin basic protein (MBP) peptide. Histologically, both the active and adoptive forms of the disease are characterized by extensive mononuclear cell infiltration and severe demyelination of the CNS. These results suggest that T cell responses specific for PLP139-151(S) are sufficient to induce clinical and histological R-EAE in SJL/J mice. This model should prove useful for examination of the cellular and molecular events involved in clinical relapses and perhaps in determining the role of PLP-specific T cell responses in multiple sclerosis (MS).

The effects of neuroantigen-specific tolerance on the induction and effector stages of EAE were examined. Tolerance induced by the i.v. injection of syngeneic splenocytes coupled with purified neuroantigens or encephalitogenic peptides of MBP and PLP using ethylene carbodiimide was extremely effective in both prevention and treatment of acute and relapsing forms of EAE in Lewis rats and SJL/J mice. The unresponsiveness is rapidly-induced, dose-dependent, long-lasting, efficient, MHC class II-restricted, and exquisitely antigen-specific. This procedure targets only effector cells bearing clonotypic receptors specific for the autoantigen/autoepitope and thus does not depend upon the autoimmune response being dominated by a restricted T cell repertoire. Moreover, it does not require that the response to the autoantigen be dominated by recognition of a specific epitope(s) within a particular autoantigen, or even the identification of the specific autoantigen. The results also demonstrate the usefulness of peripheral tolerance induced by antigen-coupled syngeneic splenocytes for identifying the fine specificity of autoimmune T cell responses which appear to change during the progression of relapsing EAE. Thus, this technique offers major advantages over many other currently employed immunoregulatory strategies and is therefore relevant for establishment of therapeutic protocols for the antigen-specific treatment of human T cell-dependent autoimmune disorders.

Natural killer cell activity was compared in the Long-Sleep and Short-Sleep mouse lines. These mice, initially selected for their sensitivities to a hypnotic dose of ethanol, are also differentially sensitive to other agents which act through the benzodiazepine/GABA receptor chloride ionophore complex. Natural killer cell activity was 40-59% lower in Short-Sleep when compared to Long-Sleep mice. Flow cytofluorometric analysis demonstrated that the number of Nk-1+ cells was also lower in the spleens of Short-Sleep than Long-Sleep mice. In addition, the incidence of 3-methylcholanthrene-induced tumors was significantly greater in Short-Sleep (85.7%) than in Long-Sleep (14.3%) mice. These results suggest that the Long-Sleep and Short-Sleep mouse lines may represent a unique model to assess the physiological role of the benzodiazepine/GABA receptor chloride ionophore complex in the neural modulation of immune function.

The possibility that sexual behavior is associated with changes in natural killer cell activity was explored using a 4-h chromium-51 release assay. Mated male Golden Hamsters (Mesocricetus auratus) showed a significant suppression of natural killer cell activity 2 h after sexual activity relative to matched Virgin controls. Natural killer cell activity returned to control levels by 16 h postmating. The suppression of natural killer cell activity did not correlate with changes in plasma cortisol, transcortin, or testosterone levels, or with ejaculatory or intromissive behaviors. Administration of 0.40 mg/100 g of testosterone also suppressed natural killer activity at 2 h.