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LymphohistiocytosisDisorder HemophagocyticCells To Control the Immunoregulatory Etoposide Selectively Ablates Activated T

JordanJonathan D. Katz, David A. Hildeman and Michael B. Theodore S. Johnson, Catherine E. Terrell, Scott H. Millen,

http://www.jimmunol.org/content/192/1/84doi: 10.4049/jimmunol.1302282November 2013;

2014; 192:84-91; Prepublished online 20J Immunol 

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2.DC1http://www.jimmunol.org/content/suppl/2013/11/20/jimmunol.130228

Referenceshttp://www.jimmunol.org/content/192/1/84.full#ref-list-1

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The Journal of Immunology

Etoposide Selectively Ablates Activated T Cells To Controlthe Immunoregulatory Disorder HemophagocyticLymphohistiocytosis

Theodore S. Johnson,* Catherine E. Terrell,† Scott H. Millen,† Jonathan D. Katz,†

David A. Hildeman,† and Michael B. Jordan†,‡

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. The Journal of Immunology, 2014, 192: 84–91.

Although immunity against pathogens is necessary forsurvival, the specificity and magnitude of immune re-sponses must be tightly regulated to avoid dangerous

immunopathology. Hemophagocytic lymphohistiocytosis (HLH) isa unique inborn disorder of immune regulation in which immuneresponses are appropriately directed (not autoimmune) but may berapidly fatal as the result of inefficient control of T cell activation(1). Mutations affecting prf1 (perforin) and related genes were foundto be causal in most affected families. Studies in animal modelsestablished a chain of pathogenesis in which defective perforin-mediated cytotoxic feedback allows prolonged/excessive Ag pre-sentation by dendritic cells (DCs), which drives abnormal CD8+

T cell activation, IFN-g production, and subsequent macrophage-mediated immunopathology (2–5). Although this pathologic im-mune activation may have varied manifestations, a constellation offeatures typifies patients with HLH: fever, splenomegaly, severecytopenias, elevated ferritin (often extreme), depressed levels offibrinogen (paradoxical, in the context of inflammation), elevatedmarkers of T cell and macrophage activation (e.g., soluble CD25 andCD163), and hemophagocytosis (the appearance of macrophagesengulfing blood and marrow cells) in various tissues (6, 7).Although HLH is rapidly fatal without specific therapy, treat-

ment with the chemotherapeutic agent etoposide (ETOP) wasempirically discovered to be life-saving .30 years ago (8). In-ternational follow-up studies established ETOP-based regimens asthe standard of care (9, 10). Despite this extensive clinical study, itremains unclear how ETOP treats HLH or why it has succeededwhile other chemotherapeutic or immunosuppressive agents triedsporadically in the 1970s and 1980s were largely unsuccessful(11). The only study (12) to examine this question analyzedETOP-driven apoptosis of lymphocytes from patients with HLHand found that they responded in a similar fashion as did thosefrom normal individuals. The question of how a chemotherapeuticagent treats an intrinsic immune regulatory disorder is alsobroadly relevant to the fields of autoimmunity and transplantation,in which DNA-damaging chemotherapeutic agents are used totreat rheumatoid arthritis, systemic lupus erythematosus, andmultiple sclerosis, as well as to prevent graft-versus-host disease.In the current study, we used a well-described murine model of

HLH, involving lymphocytic choriomeningitis virus (LCMV) in-fection of perforin-deficient (prf2/2) mice, to study the in vivotherapeutic mechanism of action for ETOP. We found that ETOPhas clear therapeutic activity in this model, alleviating all featuresof HLH. When compared with a broad array of chemotherapeuticagents, we found that it was relatively unique. Because ETOP is

*Cancer Immunology, Inflammation and Tolerance Program, Division of PediatricHematology/Oncology, Department of Pediatrics, Georgia Regents University Can-cer Center, Georgia Regents University, Augusta, GA 30912; †Division of Immu-nobiology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center,University of Cincinnati College of Medicine, Cincinnati, OH 45229; and‡Division of Bone Marrow Transplantation and Immune Deficiency, Department ofPediatrics, Cincinnati Children’s Hospital Medical Center, University of CincinnatiCollege of Medicine, Cincinnati, OH 45229

Received for publication August 27, 2013. Accepted for publication October 19,2013.

This work was supported by National Institutes of Health Grant R01HL091769 andthe Primary Immune Deficiency Treatment Consortium as a subaward from NationalInstitutes of Health Grant U54 AI082973.

Address correspondence and reprint requests to Dr. Michael B. Jordan, CincinnatiChildren’s Hospital Medical Center, ML7038, 3333 Burnet Avenue, Cincinnati, OH45229. E-mail address: michael.jordan@cchmc.org

The online version of this article contains supplemental material.

Abbreviations used in this article: CDDP, cisplatin; CLO, clofarabine; CPM, cyclo-phosphamide; DC, dendritic cell; DEX, dexamethasone; DKO, double knockout;DOXO, doxorubicin; ETOP, etoposide; FLU, fludarabine; HLH, hemophagocyticlymphohistiocytosis; LCMV, lymphocytic choriomeningitis virus; MTX, methotrex-ate; PS, phosphatidylserine; WT, wild-type.

Copyright� 2013 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302282

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often observed to have rapid and dramatic effects in clinical HLH,it has been commonly speculated that it may act directly oninflamed macrophages. Contrary to this expectation, we found nodirect beneficial effect on either macrophage activation or Agpresentation by DCs. Instead, we found that ETOP strongly sup-pressed IFN-g levels. This, in turn, was due to near completeablation of pathologically activated, virus-specific T cells. Nota-bly, this depletion was also seen in wild-type (WT) animals andwas extremely specific in vivo; quiescent naive and memory cellswere largely spared. Because of its potency and selectivity, weconclude that ETOP has unique immunomodulating qualities inHLH and may have broader usefulness as an immunosuppressiveagent.

Materials and MethodsMice, viruses, and in vivo treatments

C57BL/6, prf2/2, and IFN-g2/2 mice were obtained from The JacksonLaboratory. TCR-transgenic P14, SMARTA, and OT1 mice were a giftfrom P. Marrack (University of Colorado/Howard Hughes Medical Insti-tute, Denver, CO). Mice were housed in a specific pathogen–free facility,and animal experiments were approved by the Institutional Animal Careand Use Committee of Cincinnati Children’s Hospital Medical Center.LCMV-WE viral stocks were generated and titered as described (13). Micewere infected via i.p. injection of 200 PFU. Vaccinia-ova was obtainedfrom P. Marrack, and 105 PFU was injected i.v. All chemotherapeuticswere obtained from the Cincinnati Children’s Hospital Medical Centerclinical pharmacy: ETOP (50 mg/kg) (14), cyclophosphamide (CPM; 100mg/kg) (15), methotrexate (MTX; 70 mg/kg) (16), cisplatin (CDDP; 10mg/kg) (17), clofarabine (CLO; 8 mg/kg) (18, 19), dexamethasone (DEX;3 mg/kg) (20), doxorubicin (DOXO; 1 mg/kg) (21), fludarabine (FLU; 300mg/kg) (22), 5-fluorouracil (20 mg/kg) (23), and vinblastine (0.5 mg/kg)(24). They were administered as single doses via i.p. injection after dilu-tion or reconstitution in saline or 5% dextrose. ETOP carrier (65% PEG300, 30% ethanol, 5% Tween 80; 50 mcl/mouse) was used as indicated.Mice were examined longitudinally, typically three times/wk, for devel-opment of signs of HLH-like disease using a clinical scoring system to gaugedisease severity (Supplemental Table I). For IFN-g infusion, osmotic pumps(Durect) were loaded with IFN-g and implanted s.c. as previously described (4).

ELISA and complete blood counts

ELISAs were performed on either serum (IFN-g) or plasma (soluble CD25,ferritin, fibrinogen). Commercially available ELISA kits were used toassess fibrinogen and ferritin levels, and assays were performed accordingto the manufacturer’s instructions (Innovative Research). For other ELI-SAs, capture and detecting Abs were purified from hybridoma supernatantsas previously described (3,4). Complete blood counts were obtained usinga dedicated veterinary machine (Hemavet950FS; Drew Scientific).

Cell isolation, adoptive transfers, and cell culture

For in vivo generation of quiescent memory T cells, 3000 CD45.1+ OT1T cells were transferred into naive prf2/2 mice 1 d prior to vaccinia-OVAinfection. In vitro ETOP cultures were as follows: spleens cells from un-infected or LCMV-infected mice were purified using Ficoll and then cul-tured overnight in DMEM with either IL-7 (0.5 ng/ml; PeproTech) or IL-2(100 U/ml, human clinical grade) for cells from naive or LCMV-infectedmice, respectively. After overnight culture, cells were purified again usingFicoll and cultured with ETOP for 4 h. The drug was washed out, and cellswere cultured overnight in cytokine and then assessed for apoptosis 12 hafter ETOP pulse. For ex vivo Ag-presentation assays, DCs were isolatedusing magnetic beads (70–80% CD11c+/MHC II+; mostly CD11b+ afterLCMV) and cultured with LCMV-specific T cells (P14 or SMARTA) asdescribed (25). Supernatants were harvested after 24 h for analysis of IFN-g production by the T cells in response to Ag presentation.

Flow cytometry and microscopy

All Abs were obtained from BioLegend or eBioscience. Tetrameric MHCreagents were produced in SF9 insect cells as described (26). For quanti-tation of T cells producing IFN-g in vivo, mice were injected with bre-feldin A prior to sacrifice, and spleens were crushed in PBS/1%paraformaldehyde as described (3). For measuring phosphatidylserine (PS)exposure, cells were stained with recombinant Alexa Fluor 647–labeledMFG-E8-D89E, a high affinity PS-binding protein (27). For microscopy,

tissues were fixed in 10% buffered formalin and embedded in paraffin,followed by sectioning and staining with H&E. Images were obtained withan Evos XL core microscope using a 103 or 203 objective.

Statistics

All statistical tests were performed using NCSS2007 statistical analysissoftware. Where appropriate, results are given as the mean 6 SEM, withstatistical significance determined by a two-tailed t test.

ResultsETOP treatment rescues LCMV-infected prf2/2 mice fromHLH-like disease

LCMV-infected prf2/2 mice were previously characterized by ourgroup and validated as a robust murine model for human HLH(2). These mice develop a fatal HLH-like syndrome within weeksfollowing LCMV infection, displaying all of the diagnostic andtypical features seen in patients with HLH. We tested the effects ofETOP on HLH-like disease development in this model by givinga single i.p. injection (50 mg/kg) at 5 d post-LCMV infection. Wechose this time point because it is around the time that immuneresponses in WT and prf2/2 mice begin to diverge, and we chosethis dose because it is within the range of xenograft studies and isthe murine equivalent of what is used clinically for treating HLH(1, 28, 29). We found that prf2/2 mice treated with ETOP survivedlong-term after LCMV infection, whereas those treated with drugvehicle died within 3 wk of infection (Fig. 1A). A dose titrationrevealed that ETOP’s effects on survival and immune activation(see below) were titratable (Supplemental Fig. 1). To more ac-curately measure global disease severity in LCMV-infected mice,we developed a clinical scoring system, ranging from asymp-tomatic (0) to dead (12), based on weight loss, stance, coordina-tion, skin tenting, conjunctivitis, ascites, and mortality (SupplementalTable I). As expected, LCMV-infected prf2/2 mice displayedmuch higher clinical scores, whereas LCMV-infected WT miceexperienced mild clinical symptoms and generally recovered byday 20 of illness (Fig. 1B). ETOP-treated prf2/2 mice had higherinitial scores than did WT mice, but they recovered and displayedprolonged survival (Fig. 1A). ETOP-treated WT mice displayedno clear benefit or worsening using this scale compared with WTcontrols (data not shown).We also examined HLH disease-related biomarkers and cyto-

penias in these mice. In human HLH, elevations in plasma-solubleCD25 (IL-2R, a-chain) and ferritin levels are thought to correlatewith systemic T cell activation and acute-phase inflammation,respectively, whereas variable decreases in plasma fibrinogenlevels are less well understood (7). These biomarker profiles arerecapitulated in LCMV-infected prf2/2 mice relative to infectedWT controls, but they were efficiently reversed with ETOP ad-ministration (Fig. 1C–E). Furthermore, ETOP treatment rescuedLCMV-infected prf2/2 mice from the development of cytopenias(Fig. 1F–H). Finally, we examined multiple tissues for evidence ofhistologic improvement and found that ETOP preserved normalsplenic architecture (which is largely effaced in prf2/2 mice afterLCMV infection), decreased inflammatory liver infiltrates, and,ironically, increased marrow cellularity (Fig. 1I). Thus, ETOPtreatment leads to improvements in disease severity, mortality,disease-related biomarkers, cytopenias, and histology in LCMV-infected prf2/2 mice.

Only select chemotherapeutic agents have efficacy for treatingmurine HLH

We next sought to determine whether ETOP is unique in its abilityto treat murine HLH or whether this is a general attribute of cy-totoxic chemotherapy. Fig. 2 shows the clinical score (Fig. 2A,

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2B) and blood hemoglobin levels (Fig. 2C) for LCMV-infectedprf2/2 mice given a single i.p. injection of various chemotherapydrugs 5 d post-LCMV infection. For the sake of clarity, drugs thatexhibited therapeutic activity are shown in Fig. 2A, whereas thosethat failed to improve the disease course are shown in Fig. 2B. Aswas seen with ETOP, mice treated with either CPM or MTXsurvived (Fig. 2A). The kinetics of their response after treatment

with CPM or MTX and reversal of HLH-related anemia (Fig. 2C)were similar to that seen with ETOP. In contrast, no improvementin the disease course was demonstrated in LCMV-infected prf2/2

mice treated with other cytolytic or anti-inflammatory agents us-ing published murine doses (Fig. 2B, 2C). Attempts at dose es-calation (CDDP, CLO, DOXO, FLU) resulted in early mortality,and attempts at serial dosing on a daily schedule (DEX, FLU) did

FIGURE 1. ETOP treatment rescues LCMV-

infected prf2/2 mice from HLH-like disease.

LCMV-infected prf2/2 mice were treated with

ETOP or carrier by i.p. injection 5 d postinfec-

tion. LCMV-infected WT mice treated with

carrier 5 d postinfection are included for com-

parison. Mice were monitored longitudinally

for survival (A) and disease severity using

a clinical scoring system described inMaterials

and Methods (B). Plasma was assessed by

ELISA at day 8 postinfection for soluble CD25

(C) and at day 12 for ferritin (D), and fibrinogen

(E). Blood was drawn on day 15 d after LCMV

infection for assessment of blood counts, in-

cluding hemoglobin (F), platelet (G), and neu-

trophil (H) levels. (I) Tissues were obtained 12 d

postinfection and stained with H&E, revealing

the preservation of normal splenic architecture,

decreased liver infiltrates, and improved mar-

row cellularity with ETOP treatment. Scale bar,

100 mm. Data are mean 6 SEM and are com-

piled from 8–23 mice/group assayed across

three or more experiments. 1All mice in group

deceased. *p , 0.005.

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not alter the disease course (data not shown). Thus, ETOP appearsrelatively unique among a range of agents tested for its ability toalleviate murine HLH. The two exceptions to this finding, CPMand MTX, are notable for their uses as immune suppressive agentsin a variety of clinical contexts or for intrathecal treatment ofHLH-associated inflammation of the CNS.

ETOP treatment does not directly decrease macrophageactivation or Ag presentation by DCs in LCMV-infected prf2/2

mice

Based on clinical observations and more recent experimental data(4), it is widely believed that systemic activation of macrophagesplays a prominent role in the development of HLH. This fact,combined with the prompt clinical response observed in somepatients after starting ETOP, suggested to many that ETOP exertsits therapeutic effect by suppressing macrophage activation and/orAg presentation (30). To assess this potential mechanism of ac-tion, we examined macrophage phenotypes after ETOP treatmentof LCMV-infected prf2/2 mice. When we examined tissues 1–2wk after ETOP treatment, we observed decreased macrophageinfiltration by histology and flow cytometry (F4/80+ cells de-creased from ∼13 to 9%) (Fig. 1, data not shown). However, be-

cause disease development was completely reversed in theseanimals, it was not clear whether this was simply a secondaryeffect. In contrast, when we examined tissues on day 7, aftertreating with ETOP on day 5, we found that the highly activatedphenotype of splenic macrophages (assessing CD40, CD80,CD86, and MHC class II on F4/80+ cells in spleen, marrow, andliver) was unchanged (Fig. 3A, 3B, data not shown). Furthermore,the numbers of macrophages and DCs, as a percentage ofsplenocytes, were not decreased after ETOP treatment (∼13% F4/80+

and 2% CD11c+/MHC II+, with or without treatment). In addi-tion to surface phenotype and numbers, we sought to assess thefunction of APCs after ETOP. We recently reported that abnor-malities of DC function, rather than DC numbers, are a criticaldeterminant of immune dysregulation in prf2/2 mice (5). To in-vestigate this, we isolated DCs from ETOP- or carrier-treated,LCMV-infected prf2/2 mice (treatment on day 5, DC isolationon day 7) and cultured them with LCMV-specific CD8+ and CD4+

T cells. As expected, we found that DCs from prf2/2 mice pre-sented endogenously derived viral Ag much more potently thandid those from WT mice (Fig. 3C, 3D). Notably, DCs from ETOP-treated prf2/2 mice revealed no decrease in Ag-presentation po-tency. In fact, in some experiments, Ag presentation was actuallyincreased by ETOP treatment (Fig. 3C, data not shown). Thisfinding was similar to prior observations that in vivo T cell de-pletion leads to increases in Ag presentation by DCs (5). Thus,ETOP does not appear to have a direct suppressive effect onmacrophage activation or Ag presentation by DCs.

ETOP alleviates murine HLH by suppressing IFN-g levels inLCMV-infected prf2/2 mice

Prior studies (2) indicated that hyperproduction of IFN-g is nec-essary for development of HLH in LCMV-infected prf2/2 mice.Therefore, we hypothesized that the therapeutic effect of ETOP inthese mice is due to a reduction in IFN-g levels. Accordingly, weobserved that ETOP treatment on day 5 led to a dramatic sup-pression of serum IFN-g levels in prf2/2 mice (Fig. 4A). Fur-thermore, we predicted that if suppression of IFN-g levels isa component of ETOP’s therapeutic mechanism, then drug treat-ment after the in vivo peak of cytokine production would havelittle efficacy, because macrophages have already been exposed tohigh levels of IFN-g, and ETOP does not appear to act directly onmacrophages (2, 4). In contrast, if ETOP directly affected mac-rophage activation, then treatment would still be therapeutic atlater time points. To test our prediction, we treated LCMV-infected prf2/2 mice with ETOP at day 10, a time point afterpeak cytokines levels but before significant anemia has developed(4). In this case, neither the disease course (Fig. 4B) nor thedisease-related anemia (Fig. 4C) was ameliorated by late ETOPtherapy. In contrast, mice deficient for both perforin and IFN-g(double knockout [DKO]) were protected from anemia and fataldisease (Fig. 4B, 4C).If decreasing inflammatory cytokine production is an important

aspect of ETOP’s mechanism in HLH, then “adding back” IFN-gto ETOP-treated prf2/2 mice would abolish its therapeutic benefit.To test this prediction, we used s.c. osmotic pumps to deliver IFN-g or saline to LCMV-infected prf2/2 mice treated with ETOP onday 5. We implanted pumps shortly after administering ETOP orcarrier, titrated to maintain serum IFN-g levels of 3–6 ng/ml for upto 7 d. This level is somewhat lower than that seen in controlLCMV-infected prf2/2 mice but much higher than that seen inETOP-treated or WT mice. Consistent with previous results, micetreated with ETOP and infused with saline showed improvementin disease symptoms and survival (Fig. 4D, 4E). However, whenmice were given exogenous IFN-g, the benefit of ETOP therapy

FIGURE 2. Only select chemotherapeutic agents have efficacy in treating

murine HLH. LCMV-infected prf2/2 mice were treated with CPM, ETOP,

MTX, cladribine (2CDA), CDDP, CLO, DOXO, FLU, 5-fluorouracil (5FU),

L-asparaginase (L-ASP), vinblastine (VBL), or carrier control (Prf2/2 CON)

by i.p. injection 5 d postinfection. LCMV-infected WT mice treated with

carrier control (WT CON) 5 d postinfection are included for comparison. Mice

were monitored serially for disease severity using a clinical scoring system to

measure therapeutic effect (A) or lack thereof (B). (C) Blood was drawn on

day 15 post-LCMV infection to assess hemoglobin levels. Data are mean 6SEM of three or four mice/group, representative of three or more independent

experiments. 1All mice in treatment group deceased. *p , 0.004.

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was completely abrogated (Fig. 4D, 4E). Thus, suppression ofexcessive IFN-g production is a key aspect of ETOP’s mechanismof action in murine HLH.

ETOP acts via selective destruction of activated T cells inLCMV-infected prf2/2 mice

Because we demonstrated previously that most detectable IFN-gin LCMV-infected prf2/2 mice is derived from highly activatedCD8+ T cells (2), we hypothesized that ETOP was suppressingexcessive IFN-g production by depleting or potentially deacti-vating these cells. To test this hypothesis directly, we measured thenumber of CD8+ T cells producing IFN-g in vivo using a previ-ously described technique (3). We found that ETOP decreased thenumber of IFN-g–producing T cells to levels that were similar to

(or lower than) those seen in WT mice (Fig. 5A). Next, we directlyassessed depletion of virus-specific T cells by staining witha peptide-MHC tetrameric staining reagent (H-2Db-Gp33) incor-porating an immunodominant LCMV epitope (Gp33–41). Wefound that ETOP treatment led to near complete ablation ofLCMV-specific CD8+ T cells in all animals (Fig. 5B). The abso-lute number of Db-Gp33+ CD8+ T cells decreased from ∼2–4million cells/spleen to ,50,000 in most animals, with almost onethird of ETOP-treated animals displaying no Db-Gp33–specificT cells above staining background. To account for the practical limitsof detection using MHC-tetrameric reagents, we (conservatively)scored animals in which no Ag-specific T cells could be detected as“100-fold” depleted. When we compiled data from multiple experi-ments involving a large number of animals (.20/group), we found

FIGURE 3. ETOP treatment does not directly

decrease macrophage activation or the quality of

Ag presentation by DCs in LCMV-infected prf2/2

mice. LCMV-infected prf2/2 mice were treated

with ETOP or carrier at 5 d postinfection. LCMV-

infected WT mice treated with carrier are included

for comparison. Surface phenotype of splenic

macrophages (F4/80+) was assessed 2 d later by

flow cytometry, including CD80 (A) and MHC

class II (B) levels. DCs (CD11c+/MHC II+) were

magnetically sorted from collagenase-treated spleens

(pooled, four animals/group) at 7 d postinfection and

plated with LCMV-specific transgenic CD8+ (C) or

CD4+ (D) T cells (P14 or SMARTA) to assess MHC

class I and class II–restricted presentation of endog-

enously acquired viral Ags. Ag presentation to

T cells was quantitated by IFN-g production after

overnight culture. No IFN-g was detected when

T cells of irrelevant specificity were cultured with

DCs (data not shown). Data are mean 6 SEM of

three or four animals/group, representative of three

independent experiments. *p , 0.05.

FIGURE 4. ETOP alleviates murine HLH by suppressing IFN-g levels in LCMV-infected prf2/2 mice. (A) LCMV-infected prf2/2 mice were treated

with carrier or ETOP 5 d postinfection, and serum was obtained on days 3, 6, 8, 10, and 13 for assessment of IFN-g levels by ELISA. LCMV-infected prf2/2

mice were treated with ETOP either before or after peak IFN-g production (day 5 or 10) and monitored longitudinally for disease severity using a clinical

scoring system (B) or were bled on day 15 to assess blood hemoglobin level (C). LCMV-infected WT mice and prf/IFN-g (DKO)-deficient mice are included

for comparison. LCMV-infected prf2/2 mice were treated on day 5 postinfection with carrier or ETOP. On day 5, these mice were also implanted with

osmotic pumps (containing either IFN-g or saline) to “add back” IFN-g that is suppressed by ETOP treatment. Pumps were calibrated to maintain serum IFN-g

levels between 3 and 6 ng/ml for up to 7 d. Mice were monitored longitudinally using a clinical scoring system (D), and blood was drawn for assessment of

hemoglobin at 15 d post-LCMV infection (E). Data are mean 6 SEM of three or four mice/group, representative of three or more experiments. 1All mice in

treatment group deceased. *p , 0.01, ETOP treatment versus carrier treatment, DKO mice versus prf2/2 mice, treatment at day 5 versus day 10, or IFN-g

infusions versus saline infusion.

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that, in both prf2/2 and WT animals, ETOP treatment causeda nearly 100-fold depletion (compared with carrier-treated micein the same experiment) of virus-specific CD8+ T cells (Fig. 5C).Thus, ETOP was suppressing IFN-g production in prf2/2 mice bypotently ablating (not “deactivating”) pathologically activated,virus-specific T cells. In contrast to this profound depletion ofLCMV-specific T cells, naive T cells (defined as CD44lo) and pre-existing (quiescent) memory T cells (see Materials and Methods)were largely spared. A similar depletional effect of ETOP wasseen on CD4+ T cells, where virus-specific cells (those stainingwith IAb-GP61 tetramer) were largely ablated, whereas naiveCD4+ T cells were spared (Fig. 5D). Although the role of CD4+

T cells in driving HLH pathogenesis is not as clear as that ofCD8+ T cells, ETOP’s ability to selectively affect activated CD8+

and CD4+ T cells in both WT and prf2/2 animals suggests thatit could be useful for immunomodulation in a broad range ofcontexts.

ETOP directly and preferentially induces apoptosis ofactivated T cells

To determine whether ETOP was acting directly on activatedT cells, we cultured spleen cells from LCMV-infected or uninfectedprf2/2 mice with a titration of drug and measured apoptosis byassessing cell permeability and PS exposure. We found that ETOPinduced apoptosis of activated T cells at physiologically relevantconcentrations after overnight culture (Fig. 6) (31). We observedsimilar results with highly purified, in vitro–activated T cells,further confirming a direct effect (data not shown). When weassessed the kinetics of in vivo therapy, we found that ETOP-induced T cell ablation was evident within 24 h of treatment,suggesting a similar mechanism (Supplemental Fig. 2). Finally,we observed that activated CD8+ T cells from LCMV-infectedmice (CD44hi, day 6) were significantly more sensitive to theapoptosis-inducing effects of ETOP than were naive T cells

(CD44lo) from uninfected animals (Fig. 6B). Thus, ETOP actsdirectly on CD8+ T cells to induce apoptosis, and activationpotentiates this effect.

DiscussionAlthough ETOP is the foundation of modern HLH therapy, therehas been little understanding of how it reverses life-threateninginflammation in patients with HLH or why it has succeeded

FIGURE 5. ETOP acts via selective de-

struction of activated effector T cells in LCMV-

infected prf2/2 mice. LCMV-infected WT or

prf2/2 mice were treated with ETOP or carrier

5 d after LCMV infection. (A) Spleens were

harvested at 8 d postinfection after in vivo

brefeldin administration for quantitation of

T cells that were producing IFN-g in vivo. (B)

In parallel experiments, day-8 spleen cells were

stained using MHC-peptide tetramers (Gp33 in

the context of Db) to delineate virus-specific

CD8+ T cells. Representative plots of live gated

spleen cells from each group are shown. The

percentage of live-gated/CD8+ cells is shown.

(C) Absolute numbers of virus-specific (Db

-GP33 tetramer+), naive (CD44lo), and quies-

cent memory CD8+ T cells were quantitated in

LCMV-infected, ETOP- or carrier-treated ani-

mals. Fold change with ETOP treatment was

calculated by comparing the total number of

each cell population in spleens of carrier- or

drug-treated animals. (D) For assessment of

quiescent memory T cells, OVA-specific T cells

primed by vaccinia-OVA infection .1 mo prior

to LCMV infection were tracked. Virus-specific

CD4+ T cells were enumerated with IAb-GP61–

80 tetramer and compared with naive CD4+

T cells in LCMV-infected, ETOP- or carrier-

treated animals. Data are mean 6 SE, with

more than eight mice/group, from three or more

experiments. *p , 0.01.

FIGURE 6. ETOP directly and preferentially induces apoptosis of ac-

tivated T cells. Spleen cells from LCMV-infected (day 6) prf2/2 mice were

cultured in the presence of ETOP or carrier for 4 h and then washed and

cultured overnight. (A) Cells were assessed for permeability (with 7-

aminoactinomycin D [7-AAD]) and PS exposure (using a recombinant PS-

binding protein, see Materials and Methods). CD8+ cells are shown. (B)

LCMV-activated (day 6, CD44hi cells) and naive (CD44lo from uninfected

mice). CD8+ cells were exposed to a titration of ETOP (or carrier) as in (A),

and apoptosis induction (percentage of PS+ cells) was measured. *p , 0.01.

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over other chemotherapeutic or anti-inflammatory agents. In thecurrent study, we found that ETOP’s therapeutic activity can berobustly reproduced in a well-established murine model of HLH.This observation gave us a unique window to dissect its mecha-nism of action in a disease-relevant context. Consistent with verysparse clinical data, we found that ETOP has a relatively uniqueability to suppress HLH-like immunopathology. Furthermore, wefound that ETOP’s effects on this pathologic immune response arehighly specific for activated T cells and largely spare quiescentT cells and innate immune cells. This high degree of selectivitysuggests that ETOP may have unique immunomodulatory quali-ties in a wider array of immunopathologic conditions. Our findingsalso underscore the potential usefulness of studying HLH asa prototype for T cell–driven disorders.Our findings have several implications related to the patho-

physiology and treatment of HLH. First, it is notable that DEX,a broadly immunosuppressive agent, had minimal therapeuticeffects compared with ETOP (Fig. 1). In additional studies (datanot shown), we found that the combination of ETOP and DEX hasadditive effects, but these benefits are modest. Because HLHappears to entail abnormal regulation of Ag presentation (5), weinterpret this finding to suggest that pharmacologic suppression ofT cell activation (such as with corticosteroids or calcineurininhibitors) may be largely futile in the presence of ongoing and/orstrong antigenic stimuli. Instead, deletion of the offending T cellsappears to be the most effective approach. Indeed, the mainclinical alternative to ETOP for treating HLH is the use of T cell–depleting Abs (antithymocyte globulin or alemtuzumab) (1).Second, although an indirect consequence of ETOP-mediatedT cell deletion, the key role of IFN-g suppression underscoresthe importance of this inflammatory mediator in HLH and pro-vides additional impetus for translational efforts testing IFN-gblockade. One caveat: the relatively narrow time window that weobserved for effective ETOP treatment suggests that kinetics orother aspects (perhaps IFN-g related) of this model diverge fromclinical HLH, in which ETOP may be effective even after pro-longed illness. Third, our findings underscore the importance ofT cells as therapeutic targets in HLH, reinforcing existing clinicaland experimental data.First synthesized in 1966, ETOP exhibits antineoplastic activity

against acute myeloid leukemia, lymphomas, and a variety ofsolid-tumor cancers (32). ETOP inhibits topoisomerase II, leadingto dsDNA breaks (32), although it is not precisely understood howthis specific genotoxic lesion exerts a selective effect on activatedT cells. Ongoing studies on this topic may reveal new aspects ofT cell biology and new therapeutic targets for immunologic dis-eases.Of the agents tested, three chemotherapeutic drugs were capable

of attenuating murine HLH disease pathology: ETOP, CPM, andMTX. Although these agents are biochemically diverse (a topo-isomerase inhibitor, DNA alkylator, and an antimetabolite, re-spectively), they share clinical usefulness as immunosuppressivedrugs. Although ETOP is the basis of modern HLH therapy, MTXis used as intrathecal therapy for CNS inflammation in HLH (6, 33),whereas CPM rarely has been reported as monotherapy (34) or aspart of combination chemotherapy (35) of HLH. Although ETOPhas not been studied in the context of auto- or alloimmunity, bothMTX and CPM are used in a variety of severe autoimmune con-ditions and for the prevention/treatment of graft-versus-host dis-ease (36, 37). Surprisingly, FLU, a purine analog with strongantilymphocyte qualities (38), was not therapeutic in our system,despite multiple dosing strategies. Therefore, it appears that mecha-nistic differences between these agents are relevant to their immu-nomodulatory qualities and will require further study.

One of the most notable findings of the current study is theremarkable selectivity that we observed for ETOP as a T cell–depleting and immunomodulating agent. It ablated activated, an-tiviral T cells nearly 100-fold and (indirectly) suppressed in-flammatory cytokine levels, while essentially sparing quiescentnaive and memory T cells. Our findings are consistent with priorreports noting that ETOP causes apoptosis of activated, but notresting, lymphocytes in vitro (39–41). Notably, we observedsimilar potency and selectivity in prf2/2 and WT animals (Fig. 5,data not shown). Together, these findings suggest that ETOP couldbe used to efficiently eliminate unwanted or pathologic T cells ina wider variety of contexts, based on their activation status. In-deed, as detailed by McNally et al. (42) in this issue, etoposideappears to have similarly immune selective therapeutic effect inexperimental autoimmune encephalitis. Sparing of quiescentT cells would preserve protective memory responses and wouldnot impair responses to newly encountered pathogens. However,additional studies are needed because other investigators observedtemporary suppressive effects on T cells surviving ETOP exposure(43). Despite potential limits, a selective deletional approachcould compare very favorably with nonselective T cell–depletingagents (such as alemtuzumab) or broadly immunosuppressivedrugs (such as corticosteroids). However, additional preclinicalstudies will be needed to assess the potential efficacy of ETOP inimmune disorders beyond HLH.The principal adverse effects of ETOP and other chemothera-

peutic agents relate to off-target genotoxicity, including dose-dependent suppression of hematopoiesis and risk for secondaryleukemia. In general, therapy-related leukemia has been reportedafter treatment with prolonged or high-intensity combinationregimens incorporating ETOP and other genotoxic agents, althoughpatients with HLH receiving unusually prolonged courses of ETOPmonotherapy have developed leukemia (44, 45). Paradoxically, wefound that ETOP prevented the development of cytopenias andmarrow hypocellularity in treated animals, presumably because itsdesirable effects on T cells were more potent than its off-targeteffects on marrow cells. Although such adverse effects may bemanaged clinically, further studies into the mechanistic basis ofimmune modulation, genotoxicity, and marrow suppression mayhelp to further improve the therapeutic index of ETOP and relateddrugs for the treatment of HLH and other immunologic disorders.

AcknowledgmentsWe thank Dr. Erin Zoller for technical assistance in osmotic pump place-

ment and Dr. Robert Thacker for assistance with the ex vivo Ag–stimulation

assays. In addition, we appreciate the gracious support of the Cincinnati

Children’s Hospital Medical Center clinical pharmacy.

DisclosuresThe authors have no financial conflicts of interest.

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