Improvement of Refractory Systemic Juvenile Idiopathic Arthritis-Associated Lung Disease with Single-Agent Blockade of IL-1ß and IL-18.

Systemic juvenile idiopathic arthritis associated with interstitial lung disease (SJIA-LD) represents a highly morbid subset of SJIA for which effective therapies are lacking. We report the case of a patient with refractory SJIA-LD who underwent treatment with MAS-825, an investigational bispecific monoclonal antibody targeting IL-1ß and IL-18. MAS-825 treatment was associated with a marked reduction in total IL-18 and free IL-18 in both serum and bronchoalveolar lavage fluid (BAL). Baseline oxygen saturation, exercise tolerance, and quality of life metrics improved after treatment with MAS-825, while pulmonary function testing remained stable. Following treatment, the BAL showed no evidence of pulmonary alveolar proteinosis and inflammatory infiltrates were markedly reduced, reflected by decreased numbers of CD4 T-cells, CD8 T-cells, and macrophages. The patient was able to wean entirely off systemic corticosteroids and other biologics after 10 months of treatment with MAS-825 and experienced no side effects of the drug. This case demonstrates improvement in pulmonary symptoms, lung inflammation, and burden of immunomodulatory therapy after treatment with MAS-825 and suggests that simultaneous targeting of both IL-1ß and IL-18 may be a safe and effective treatment strategy in SJIA-LD.

Inherited Autoinflammatory Syndromes.

Autoinflammation describes a collection of diverse diseases caused by indiscriminate activation of the immune system in an antigen-independent manner. The rapid advancement of genetic diagnostics has allowed for the identification of a wide array of monogenic causes of autoinflammation. While the clinical picture of these syndromes is diverse, it is possible to thematically group many of these diseases under broad categories that provide insight into the mechanisms of disease and therapeutic possibilities. This review covers archetypical examples of inherited autoinflammatory diseases in five major categories: inflammasomopathy, interferonopathy, unfolded protein/cellular stress response, relopathy, and uncategorized. This framework can suggest where future work is needed to identify other genetic causes of autoinflammation, what types of diagnostics need to be developed to care for this patient population, and which options might be considered for novel therapeutic targeting.

The Effects of Gamma Irradiation on Chemical Biomarker Recovery from Mixed Chemical/Biological Threat Exposure Specimens.

BACKGROUND: Irradiative sterilization of clinical specimens prior to chemical laboratory testing provides a way to not only sterilize pathogens and ensure laboratorian safety but also preserve sample volume and maintain compatibility with quantitative chemical diagnostic protocols. Since the compatibility of clinical biomarkers with gamma irradiation is not well characterized, a subset of diagnostic biomarkers ranging in molecular size, concentration, and clinical matrix was analyzed to determine recovery following gamma irradiation. METHODS: Sample irradiation of previously characterized quality control materials (QCs) at 5 Mrad was carried out at the Gamma Cell Irradiation Facility at the Centers for Disease Control and Prevention (CDC) in Atlanta, GA. Following irradiation, the QCs were analyzed alongside non-irradiated QCs to determine analyte recovery between dosed and control samples. RESULTS: Biomarkers for exposure to abrin, ricin, and organophosphorus nerve agents (OPNAs) were analyzed for their stability following gamma irradiation. The diagnostic biomarkers included adducts to butyrylcholinesterase, abrine, and ricinine, respectively, and were recovered at over 90% of their initial concentration. CONCLUSIONS: The results from this pilot study support the implementation of an irradiative sterilization protocol for possible mixed-exposure samples containing both chemical and biological threat agents (mixed CBTs). Furthermore, irradiative sterilization significantly reduces a laboratorian's risk of infection from exposure to an infectious agent without compromising chemical diagnostic testing integrity, particularly for diagnostic assays in which the chemical analyte has been shown to be fully conserved following a 5 Mrad irradiative dose.

Genetic Deficiency of Interferon-γ Reveals Interferon-γ-Independent Manifestations of Murine Hemophagocytic Lymphohistiocytosis.

OBJECTIVE: Familial hemophagocytic lymphohistiocytosis (FHLH) is a complex cytokine storm syndrome caused by genetic abnormalities rendering CD8+ T cells and natural killer cells incapable of cytolytic killing. In murine models of FHLH, interferon-γ (IFNγ) produced by CD8+ T cells has been identified as a critical mediator of disease, and an IFNγ-blocking antibody (emapalumab) has recently been approved by the Food and Drug Administration. However, development of hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) in patients who are genetically unresponsive to IFNγ questions the absolute necessity of IFNγ in driving disease. This study was undertaken to determine the necessity of IFNγ in driving HLH. METHODS: IFNγ-/- Prf1-/- mice were infected with lymphocytic choriomeningitis virus (LCMV), and HLH immunopathologic features, including survival, weight loss, cytopenias, cytokine profiles, and immune cell phenotypes, were assessed. Mixed bone marrow chimeras were created to determine the immune cell-intrinsic role of IFNγ receptor signaling. CD8+ T cell depletion and interleukin-33 (IL-33)/ST2 blockade were performed using monoclonal antibodies. RESULTS: LCMV infection of IFNγ-/- Prf1-/- mice resulted in severe HLH-like disease. CD8+ T cells and the IL-33/ST2 axis remained essential mediators of disease; however, IFNγ-independent HLH immunopathology correlated with a 10-15-fold increase in neutrophilia (P < 0.001) and an altered cytokine milieu dominated by IL-6, IL-1ß, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05). Furthermore, IFNγ regulated CD8+ T cell expression of GM-CSF and neutrophil survival. CONCLUSION: IFNγ is not necessary for the development of fulminant HLH, requiring physicians to consider case-by-case treatment strategies. Use of therapies that target upstream activators of CD8+ T cells, such as IL-33/ST2 signaling, may be more universally applicable treatment options that ameliorate both IFNγ-dependent and -independent manifestations of HLH/MAS.

Disruption of IL-33 Signaling Limits Early CD8+ T Cell Effector Function Leading to Exhaustion in Murine Hemophagocytic Lymphohistiocytosis.

Danger signals mediated through ST2, the interleukin-33 (IL-33) receptor, amplify CD8+ T cell-mediated inflammation in the murine model of familial hemophagocytic lymphohistiocytosis type 2 (FHL2), and blockade of ST2 provides a potential therapeutic strategy in this disease. However, the long-term effects of disrupting IL-33/ST2 signaling on the CD8+ T cell compartment are unknown. Here, we examined the evolution of the T cell response in murine FHL type 2 in the absence of ST2 signaling and found that CD8+ T cells gradually undergo exhaustion, similar to a related nonfatal FHL model. ST2 inhibition indirectly promotes CD8+ T cell exhaustion, and in contrast to other forms of FHL, reversal of exhaustion does not affect mortality. Disruption of IL-33 signaling exerts a more significant impact on the CD8+ T cell compartment early in the course of disease by intrinsically limiting CD8+ T cell proliferative and cytokine production capacity. Our data thus suggest that while ST2 blockade ultimately enables the development of CD8+ T cell exhaustion in late-stage murine FHL2, exhaustion is merely an effect, rather than the cause, of extended survival in these mice. The acute impact of ST2 inhibition on both the quantity and quality of the effector CD8+ T cell response more likely underlies the protective benefits of this treatment. This study provides evidence that redefines the relationship between CD8+ T cell exhaustion and mortality in murine FHL and supports the therapeutic use of ST2 blockade during the acute stage of disease.

Muscularis macrophage development in the absence of an enteric nervous system.

The nervous system of the bowel regulates the inflammatory phenotype of tissue resident muscularis macrophages (MM), and in adult mice, enteric neurons are the main local source of colony stimulating factor 1 (CSF1), a protein required for MM survival. Surprisingly, we find that during development MM colonize the bowel before enteric neurons. This calls into question the requirement for neuron-derived CSF1 for MM colonization of the bowel. To determine if intestinal innervation is required for MM development, we analyzed MM of neonatal Ret-/- (Ret KO) mice that have no enteric nervous system in small bowel or colon. We found normal numbers of well-patterned MM in Ret KO bowel. Similarly, the abundance and distribution of MM in aganglionic human colon obtained from Hirschsprung disease patients was normal. We also identify endothelial cells and interstitial cells of Cajal as the main sources of CSF1 in the developing bowel. Additionally, MM from neonatal Ret KOs do not differ from controls in baseline activation status or cytokine-production in response to lipopolysaccharide. Unexpectedly, these data demonstrate that the enteric nervous system is dispensable for MM colonization and patterning in the bowel, and suggest that modulatory interactions between MM and the bowel nervous system are established postnatally.

IL-10 distinguishes a unique population of activated, effector-like CD8+ T cells in murine acute liver inflammation.

Immune-mediated liver injury is a central feature of hyperinflammatory diseases, such as hemophagocytic syndromes, yet the immunologic mechanisms underlying those processes are incompletely understood. In this study, we used the toll-like receptor 9 (TLR9)-mediated model of a hemophagocytic syndrome known as macrophage activation syndrome (MAS) to dissect the predominant immune cell populations infiltrating the liver during inflammation. We identified CD8+ T cells that unexpectedly produce interleukin-10 (IL-10) in addition to interferon-γ (IFN-γ) as a major hepatic population induced by TLR9 stimulation. Despite their ability to produce this anti-inflammatory cytokine, IL-10+ hepatic CD8+ T cells in TLR9-MAS mice did not resemble CD8+ T suppressor cells. Instead, the induction of these cells occurred independently of antigen stimulation and was partially dependent on IFN-γ. IL-10+ hepatic CD8+ T cells demonstrated an activated phenotype and high turnover rate, consistent with an effector-like identity. Transcriptional analysis of this population confirmed a gene signature of effector CD8+ T cells yet suggested responsiveness to liver injury-associated growth factors. Together, these findings suggest that IL-10+ CD8+ T cells induced by systemic inflammation to infiltrate the liver have initiated an inflammatory, rather than regulatory, program and may thus have a pathogenic role in severe, acute hepatitis.

ST2 contributes to T-cell hyperactivation and fatal hemophagocytic lymphohistiocytosis in mice.

Cytokine storm syndromes, such as familial hemophagocytic lymphohistiocytosis (FHL), are lethal disorders caused by uncontrolled, systemic immune activation. In the murine model of FHL, in which perforin-deficient (Prf1(-/-)) mice are infected with lymphocytic choriomeningitis virus (LCMV), disease is driven by overabundant interferon (IFN)γ-producing LCMV-specific CD8(+) T cells thought to arise from excessive antigen stimulation through the T-cell receptor. However, this paradigm is insufficient to explain several fundamental aspects of FHL, namely, the inability of many pathogenic antigens to induce hyperinflammation, and the previously identified role of MyD88 in the disease. We now show a novel role for the MyD88-dependent interleukin-33 (IL-33) receptor, ST2, in FHL. Expression of IL-33 and ST2 is upregulated in LCMV-infected Prf1(-/-) mice. Blockade of ST2 markedly improves survival of LCMV-infected Prf1(-/-) mice and reduces the severity of multiple disease parameters, including serum levels of IFNγ. This decrease in IFNγ corresponds to a reduction in both the frequency of IFNγ(+) LCMV-specific CD8(+) and CD4(+) T cells and the magnitude of IFNγ expression in these cells. These findings demonstrate that disruption of ST2 signaling in the murine model of FHL reduces T cell-mediated production of IFNγ and suggest a revised paradigm in which danger signals such as IL-33 are crucial amplifiers of immune dysregulation in FHL. Furthermore, this study provides evidence to support blockade of ST2 as a novel therapeutic strategy for FHL.

Cytotoxic capacity of SIV-specific CD8(+) T cells against primary autologous targets correlates with immune control in SIV-infected rhesus macaques.

Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8(+) T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4(+) T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+) T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], p = 0.002). In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed. Furthermore, the CD8(+) T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004). These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.

Trivalent adenovirus type 5 HIV recombinant vaccine primes for modest cytotoxic capacity that is greatest in humans with protective HLA class I alleles.

If future HIV vaccine design strategies are to succeed, improved understanding of the mechanisms underlying protection from infection or immune control over HIV replication remains essential. Increased cytotoxic capacity of HIV-specific CD8+ T-cells associated with efficient elimination of HIV-infected CD4+ T-cell targets has been shown to distinguish long-term nonprogressors (LTNP), patients with durable control over HIV replication, from those experiencing progressive disease. Here, measurements of granzyme B target cell activity and HIV-1-infected CD4+ T-cell elimination were applied for the first time to identify antiviral activities in recipients of a replication incompetent adenovirus serotype 5 (Ad5) HIV-1 recombinant vaccine and were compared with HIV-negative individuals and chronically infected patients, including a group of LTNP. We observed readily detectable HIV-specific CD8+ T-cell recall cytotoxic responses in vaccinees at a median of 331 days following the last immunization. The magnitude of these responses was not related to the number of vaccinations, nor did it correlate with the percentages of cytokine-secreting T-cells determined by ICS assays. Although the recall cytotoxic capacity of the CD8+ T-cells of the vaccinee group was significantly less than that of LTNP and overlapped with that of progressors, we observed significantly higher cytotoxic responses in vaccine recipients carrying the HLA class I alleles B*27, B*57 or B*58, which have been associated with immune control over HIV replication in chronic infection. These findings suggest protective HLA class I alleles might lead to better outcomes in both chronic infection and following immunization due to more efficient priming of HIV-specific CD8+ T-cell cytotoxic responses.

Defective human immunodeficiency virus-specific CD8+ T-cell polyfunctionality, proliferation, and cytotoxicity are not restored by antiretroviral therapy.

Identifying the functions of human immunodeficiency virus (HIV)-specific CD8+ T cells that are not merely modulated by the level of virus but clearly distinguish patients with immune control from those without such control is of paramount importance. Features of the HIV-specific CD8+ T-cell response in antiretroviral-treated patients (designated Rx <50) and untreated patients (long-term nonprogressors [LTNP]) matched for very low HIV RNA levels were comprehensively examined. The proliferative capacity of HIV-specific CD8+ T cells was not restored in Rx <50 to the level observed in LTNP, even though HIV-specific CD4+ T-cell proliferation in the two patient groups was comparable. This diminished HIV-specific CD8+ T-cell proliferation in Rx <50 was primarily due to a smaller fraction of antigen-specific cells recruited to divide and not to the numbers of divisions that proliferating cells had undergone. Exogenous interleukin-2 (IL-2) induced proliferating cells to divide further but did not rescue the majority of antigen-specific cells with defective proliferation. In addition, differences in HIV-specific CD8+ T-cell proliferation could not be attributed to differences in cellular subsets bearing a memory phenotype, IL-2 production, or PD-1 expression. Although polyfunctionality of HIV-specific CD8+ T cells in Rx <50 was not restored to the levels observed in LTNP despite prolonged suppression of HIV RNA levels, per-cell cytotoxic capacity was the functional feature that most clearly distinguished the cells of LTNP from those of Rx <50. Taken together, these data suggest that there are selective qualitative abnormalities within the HIV-specific CD8+ T-cell compartment that persist under conditions of low levels of antigen.

Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control.

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.