Catherine E. Terrell

Hemophagocytosis causes a consumptive anemia of inflammation

IFN-γ stimulates blood-eating macrophages (hemophagocytes) by acting directly on macrophages to promote phagocytosis and uptake of blood cells.

Perforin is a critical physiologic regulator of T-cell activation

Individuals with impaired perforin-dependent cytotoxic function (Ctx(-)) develop a fatal inflammatory disorder called hemophagocytic lymphohistiocytosis (HLH). It has been hypothesized that immune hyperactivation during HLH is caused by heightened infection, defective apoptosis/responsiveness of Ctx(-) lymphocytes, or enhanced antigen presentation. Whereas clinical and experimental data suggest that increased T-cell activation drives HLH, potential abnormalities of T-cell activation have not been well characterized in Ctx(-) hosts. To define such abnormalities and to test these hypotheses, we assessed in vivo T-cell activation kinetics and viral loads after lymphocytic choriomeningitis virus (LCMV) infection of Ctx(-) mice. We found that increased T-cell activation occurred early during infection of Ctx(-) mice, while they had viral burdens that were identical to those of WT animals, demonstrating that T-cell hyperactivation was independent of viral load. Furthermore, cell transfer and signaling studies indicated that increased antigenic stimulation, not a cell-intrinsic defect of responsiveness, underlay heightened T-cell activation in vivo. Finally, direct measurement of viral antigen presentation demonstrated an increase in Ctx(-) mice that was proportional to abnormal T-cell activation. We conclude that perforin-dependent cytotoxicity has an immunoregulatory role that is distinguishable from its pathogen clearance function and limits T-cell activation in the physiologic context by suppressing antigen presentation.

Mice with a Selective Impairment of IFN-γ Signaling in Macrophage Lineage Cells Demonstrate the Critical Role of IFN-γ–Activated Macrophages for the Control of Protozoan Parasitic Infections In Vivo

IFN-γ has long been recognized as a cytokine with potent and varied effects in the immune response. Although its effects on specific cell types have been well studied in vitro, its in vivo effects are less clearly understood because of its diverse actions on many different cell types. Although control of multiple protozoan parasites is thought to depend critically on the direct action of IFN-γ on macrophages, this premise has never been directly proven in vivo. To more directly examine the effects of IFN-γ on cells of the macrophage lineage in vivo, we generated mice called the “macrophages insensitive to IFN-γ” (MIIG) mice, which express a dominant negative mutant IFN-γ receptor in CD68+ cells: monocytes, macrophages, dendritic cells, and mast cells. Macrophage lineage cells and mast cells from these mice are unable to respond to IFN-γ, whereas other cells are able to produce and respond to this cytokine normally. When challenged in vitro, macrophages from MIIG mice were unable produce NO or kill Trypanosoma cruzi or Leishmania major after priming with IFN-γ. Furthermore, MIIG mice demonstrated impaired parasite control and heightened mortality after T. cruzi, L. major, and Toxoplasma gondii infection, despite an appropriate IFN-γ response. In contrast, MIIG mice displayed normal control of lymphocytic choriomeningitis virus, despite persistent insensitivity of macrophages to IFN-γ. Thus, the MIIG mouse formally demonstrates for the first time in vivo, the specific importance of direct, IFN-γ mediated activation of macrophages for controlling infection with multiple protozoan parasites.

Etoposide Selectively Ablates Activated T Cells To Control the Immunoregulatory Disorder Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects in this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug that inhibits topoisomerase II, is the mainstay of treatment for HLH, although its therapeutic mechanism remains unknown. We used a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin-deficient mice, to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells, and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide’s immunomodulatory effects were similar in wild-type and perforin-deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have usefulness as a novel immune modulator in a broad array of immunopathologic disorders.

Mixed hematopoietic or T-cell chimerism above a minimal threshold restores perforin-dependent immune regulation in perforin-deficient mice

Defects in perforin and related genes lead to a loss of normal immune regulation and underlie hemophagocytic lymphohistiocytosis (HLH), which requires hematopoietic cell transplantation for long-term cure. However, transplantation may be complicated by the development of mixed chimerism and uncertainty regarding the risk of HLH recurrence. To help clarify this risk and investigate how perforin influences immune activation, we studied perforin-mediated immune regulation in the context of mixed chimerism using a murine model of HLH. We found that there is a distinct threshold of ∼10% to 20% perforin expression with either mixed hematopoietic or CD8(+) T cell chimerism, above which immune regulation was reestablished. These findings demonstrate that perforin-mediated immunoregulation functions in trans and are consistent with a feedback model in which cytotoxic T cells control immune activation by killing dendritic cells. These findings also suggest rational targets for maintenance of minimal posttransplant chimerism and for therapeutic strategies involving gene correction.

Eliminating Encephalitogenic T Cells without Undermining Protective Immunity

The current clinical approach for treating autoimmune diseases is to broadly blunt immune responses as a means of preventing autoimmune pathology. Among the major side effects of this strategy are depressed beneficial immunity and increased rates of infections and tumors. Using the experimental autoimmune encephalomyelitis model for human multiple sclerosis, we report a novel alternative approach for purging autoreactive T cells that spares beneficial immunity. The moderate and temporally limited use of etoposide, a topoisomerase inhibitor, to eliminate encephalitogenic T cells significantly reduces the onset and severity of experimental autoimmune encephalomyelitis, dampens cytokine production and overall pathology, while dramatically limiting the off-target effects on naive and memory adaptive immunity. Etoposide-treated mice show no or significantly ameliorated pathology with reduced antigenic spread, yet have normal T cell and T-dependent B cell responses to de novo antigenic challenges as well as unimpaired memory T cell responses to viral rechallenge. Thus, etoposide therapy can selectively ablate effector T cells and limit pathology in an animal model of autoimmunity while sparing protective immune responses. This strategy could lead to novel approaches for the treatment of autoimmune diseases with both enhanced efficacy and decreased treatment-associated morbidities.

Losartan for the nephropathy of sickle cell anemia: A phase-2, multicenter trial

Nephropathy is a common and progressive complication of sickle cell anemia (SCA). In SCA mice, we found that hyperangiotensinemia in the absence of hypertension underlies nephropathy, and its downregulation by losartan, an angiotensin‐II‐receptor‐1 blocker, reduced albuminuria and progression of nephropathy. Therefore, we performed a phase‐2 trial of oral losartan, given for 6 months, to explore whether it reduced albuminuria in children and adults with SCA. Participants were allocated to groups defined by class of baseline urinary albumin‐to‐creatinine ratio (UACR): no albuminuria (NoA), microalbuminuria (MicroA), and macroalbuminuria (MacroA). The primary endpoint was a ≥25% reduction UACR from baseline. There were 32 evaluable participants (mean age 24 years; NoA = 14, MicroA = 12, MacroA = 6). The primary endpoint was met in 83% of the MacroA group (P < 0.0001) and 58% of the MicroA group (P < 0.0001). Median fold‐change in UACR was −0.74 for MacroA and −0.46 for MicroA. In MacroA and MicroA, UACR classification improved in 50% but worsened in 11%. Urine osmolality and estimated glomerular filtration rate (eGFR) did not change significantly. Losartan was discontinued in three participants [leg cramps, N = 1; decline in eGFR >25% (142➝104 mL/minute/1.73 m2), N = 1; rise in serum creatinine >50% (0.2➝0.3 mg/dL), N = 1]. Albuminuria was associated with diastolic dysfunction and impaired functional capacity, although cardiopulmonary status was unchanged after 6 months of losartan therapy. In summary, losartan decreased urinary albumin excretion in most participants with albuminuria. Those with macroalbuminuria had the greatest benefit. This study forms the basis for a phase‐3, randomized, placebo‐controlled trial of losartan for the nephropathy of SCA.

237. Genetic Therapy for Perforin Deficiency Associated Hemophagocytic Lymphohistiocytosis Requires High Level Expression of the Perforin Gene for Adequate Correction

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune regulatory disorder, whereby heightened immune activation following viral infections causes hypercytokinemia, pancytopenia and end-organ damage. The majority of familial HLH is caused by Perforin-1 (prf1) gene mutations. Perforin is expressed in CD8+ T cells and natural killer (NK) cells and is critical for lymphocyte cytotoxicity. Currently, immunosuppressive therapy followed by allogeneic hematopoietic stem cell (HSC) transplant is the only curative option for HLH, but is restricted by availability of matched donors and a high (20-50%) transplant related mortality. We posited that perforin gene transfer into autologous HSC may be curative, have lower mortality, and not be limited by donor availability. We recently reported significant, but partial, correction of HLH symptoms in prf1-/- mice using lentivirus vectors (LV) expressing perforin from a ubiquitous phosphoglycerate kinase (PGK) promoter or a tissue specific perforin gene promoter (PRF). We hypothesized that negative selection of high perforin expressing HSC and the perforin gene dosage delivered by the LV contributes to the partial correction. We redesigned and developed a series of LV and compared prf1 expression from the PGK/PRF promoters to that from the MND promoter/enhancer; additionally we restricted perforin expression from PGK and MND in HSC with 4 repeats of miR126 (4T) target sequence, a miRNA specifically expressed in HSCs but not in lymphocytes, thus, circumventing the negative selection of high perforin expressing HSC. Improved perforin expression and cytotoxicity was observed with the ‘HSC-restricted’ MND4TLV in the KHYG1 human NK cell line, and in prf1-/- P14 mice, transgenic for a T-cell receptor that recognizes a lymphocytic choriomeningitis virus (LCMV) glycoprotein. These LV were compared in prf1-/- mice that were challenged with LCMV following transplant of gene-modified HSC. High gene marked NK chimerism was achieved: 87.6±1.7%, 83.1±2.4% and 58.1±4.6% with the MND4T, PGK4T and PRF0T LV. Following LCMV challenge, γ-IFN levels in MND4T, PRF0T and PGK4T mice were 1.7±0.3, 1.5±0.5 and 5.9±2.1 ng/ml, respectively, compared to 1.7±0.5 ng/ml in prf1-/- mice receiving WT HSC. Similarly, the degree of anemia in WT and MND4T mice was comparable 15 days after LCMV challenge: hemoglobins declined by 4.3±0.3, 3.7±0.3, 5.6±0.3 and 5.8±0.7 g/dL in WT, MND4T, PGK4T and PRF0T mice, respectively. Most importantly, 66.7% of the mice in the MND4T group survived the LCMV challenge, while only 10% and no mice survived in PGK4T and PRF0T groups, suggesting that perforin expression from PGK4T and PRF0T were insufficient to prevent fatal HLH in the majority of animals. Overall, we show that perforin deficient HLH requires a robust perforin expression in cytotoxic T and NK cells for adequate correction of the disease phenotype.

Correction: Eliminating Encephalitogenic T Cells without Undermining Protective Immunity

McNally, J. P., E. E. Elfers, C. E. Terrell, E. Grunblatt, D. A. Hildeman, M. B. Jordan, and J. D. Katz. 2014. Eliminating encephalitogenic T cells without undermining protective immunity. J. Immunol . 192: [73–83][1]. The e-mail address for Dr. Michael B. Jordan in the correspondence footnote