Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects of 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 which inhibits topoisomerase II, is the mainstay of treatment for HLH, though its therapeutic mechanism remains unknown. We utilized 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 utility as a novel immune modulator in a broad array of immunopathologic disorders.

Binding of pertussis toxin (PTx) was examined by glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated bi-antennary compounds with an N-glycan core terminating in α2-6 linked sialic acid. The second group consists of multi-antennary compounds with a canonical N-glycan core, but lacking terminal sialic acids, which represents a departure from previous understanding of PTx binding to N-glycans. The third group consists of Neu5Acα2-3(Lactose or N-acetyllactosamine) that lack the branched mannose core found in Nglycans, thus their presentation is more similar to O-linked glycans and glycolipids. The fourth group of compounds consists of Neu5Acα2-8Neu5Acα2-8Neu5Ac, which is seen in the c series gangliosides and some N-glycans. Quantitative analysis by SPR of the relative affinities of PTx for terminal Neu5Acα2-3 versus Neu5Acα2-6, as well as the affinities for the trisaccharide Neu5Acα2-8Neu5Acα2-8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4-to 5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds was mediated by the Cterminal binding sites, and binding to non-sialylated N-linked glycans is mediated by the Nterminal sites present on both the S2 and S3 subunits. A detailed understanding of the glycans recognized by pertussis toxin is essential to understand which cells are targeted in clinical disease.Vaccination has greatly reduced whooping cough (pertussis) morbidity and mortality; alarmingly however, the number of cases has been increasing in the US since a historic low in 1976 (1,2). Pertussis toxin (PTx) is often considered the major virulence factor of B pertussis, as PTx mutants are avirulent in mouse models, and consequently PTx is included as a component in all acellular pertussis vaccines (3). PTx alone is responsible for the systemic manifestations of lymphocytosis and hyperinsulinemia, and is the chief candidate for defense against innate and adaptive immune systems past the initial colonization (4-7).PTx is a member of the AB 5 family of bacterial toxins, which includes cholera toxin from Vibrio cholerae, heat-labile toxin from Escherichia coli, and Shiga toxin from Shigella † This work was supported by National Institutes of Health Grants R01 AI 064893 (A.A.W.) , and S5 in the ratio 1:1:2:1 (8). The A subunit, named S1 in PTx, is an ADP-ribosyltransferase which targets the α-subunit of some GTP-binding proteins (9). The B-pentamer is required for cell-targeting and cytosolic entry of S1 into mammalian cells, but also has activities independent of S1, such as antigen-independent T cell activation and mitogenicity (8,(10)(11)(12)(13)(14)(15). The fact that the binding (B) portion of the toxin has activity independent of the enzymatic acti...

Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.autoimmunity | immune regulation | DNA damage response | therapeutics T he immune system has evolved under intense pressure from pathogens that proliferate very rapidly (1). In responding to infections, the adaptive immune system needs to mobilize rare pathogen-specific lymphocytes quickly. This mobilization is achieved by rapid, exponential expansion of responding lymphocyte clones. Indeed, antigen-activated T and B cells appear to have some of the most rapid division rates among all mammalian cell types (2). We hypothesized that this unique aspect of lymphocyte biology would cause significant genomic stress in responding lymphocytes and that novel forms of therapeutic immune suppression could be developed by exploiting this weakness. Such strategies would allow for "developmental" or "kinetic" targeting of pathological immune responses at the time of disease activity or organ rejection and could complement or replace chronic suppression of immune signaling or cytokine pathways. The recent preclinical and clinical development of a wide array of rationally designed small-molecule inhibitors of various signaling and effector molecules within DNA damage-response (DDR) cascades has provided an opportunity to test this hypothesis (3, 4).We first looked for evidence of a DDR occurring in activated T cells in physiological contexts and found a broad DDR in murine and human T cells. Next, upon screening an array of DNA-damaging agents and DDR-altering small molecules for their ability to kill activated, but not resting, T cells, we identified two promising strategies. We found that inhibition of MDM2 (an end...