of July 1, 2014.This information is current as

InfectionLeishmania donovaniImmunity following

Mediated− T Cell+Long-Term CD4Tissue Requirements for Establishing

Kaye, Ashraful Haque and Christian R. EngwerdaMontes De Oca, Shannon E. Best, Kylie R. James, Paul M. Alexander, Rebecca J. Faleiro, Fiona H. Amante, MarcelaRivera, Alexander Mulherin, Meru Sheel, Clare E. Patrick T. Bunn, Amanda C. Stanley, Fabian de Labastida

http://www.jimmunol.org/content/192/8/3709doi: 10.4049/jimmunol.1300768March 2014;

2014; 192:3709-3718; Prepublished online 14J Immunol 

Referenceshttp://www.jimmunol.org/content/192/8/3709.full#ref-list-1

, 24 of which you can access for free at: cites 54 articlesThis article

Subscriptionshttp://jimmunol.org/subscriptions

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Tissue Requirements for Establishing Long-Term CD4+

T Cell–Mediated Immunity following Leishmania donovaniInfection

Patrick T. Bunn,*,†,1 Amanda C. Stanley,*,‡,1 Fabian de Labastida Rivera,*

Alexander Mulherin,*,x Meru Sheel,* Clare E. Alexander,{ Rebecca J. Faleiro,*,‖

Fiona H. Amante,* Marcela Montes De Oca,*,# Shannon E. Best,* Kylie R. James,*,#

Paul M. Kaye,** Ashraful Haque,* and Christian R. Engwerda*

Organ-specific immunity is a feature of many infectious diseases, including visceral leishmaniasis caused by Leishmania donovani.

Experimental visceral leishmaniasis in genetically susceptible mice is characterized by an acute, resolving infection in the liver

and chronic infection in the spleen. CD4+ T cell responses are critical for the establishment and maintenance of hepatic immunity

in this disease model, but their role in chronically infected spleens remains unclear. In this study, we show that dendritic cells are

critical for CD4+ T cell activation and expansion in all tissue sites examined. We found that FTY720-mediated blockade of T cell

trafficking early in infection prevented Ag-specific CD4+ T cells from appearing in lymph nodes, but not the spleen and liver,

suggesting that early CD4+ T cell priming does not occur in liver-draining lymph nodes. Extended treatment with FTY720 over

the first month of infection increased parasite burdens, although this associated with blockade of lymphocyte egress from

secondary lymphoid tissue, as well as with more generalized splenic lymphopenia. Importantly, we demonstrate that CD4+

T cells are required for the establishment and maintenance of antiparasitic immunity in the liver, as well as for immune

surveillance and suppression of parasite outgrowth in chronically infected spleens. Finally, although early CD4+ T cell priming

appeared to occur most effectively in the spleen, we unexpectedly revealed that protective CD4+ T cell–mediated hepatic immunity

could be generated in the complete absence of all secondary lymphoid tissues. The Journal of Immunology, 2014, 192: 3709–3718.

The spleen is a major secondary lymphoid organ that re-moves abnormal erythrocytes and foreign material fromthe blood. It also can play an important role in hemato-

poiesis, as well as the generation of cellular and humoral immuneresponses (reviewed in Refs. 1, 2). The importance of the spleenfor controlling blood-borne infections is evident by patients whohad undergone splenectomy being at increased risk for developingmalaria (3). In addition, individuals lacking a spleen are moresusceptible to bacterial pathogens, such as pneumococci, menin-gococci, and Haemophilus influenzae, primarily caused by an in-

ability to generate T cell–dependent Ab responses to the capsular

polysaccharides produced by these microbes (4, 5). Susceptibility

to these bacteria also arises because of the important role that the

spleen plays in removal of Ab-opsonized pathogens via well-

organized collections of macrophages that form part of the body’s

reticuloendothelial system (2).The function of the spleen to remove infectious agents from the

circulation also makes it a site of residence for many persistentviruses, bacteria, and parasites, and immune responses targeted atthese pathogens may lead to gross splenic pathology. For example,both mice and humans with visceral leishmaniasis (VL), causedby infection with the protozoan parasite Leishmania donovani,develop significant splenomegaly accompanied by disruptionsto splenic lymphoid tissue architecture and immune function(reviewed in Refs. 6, 7). Strikingly, splenic pathology develops ininfected mice concurrent with the onset of immunity directedtoward parasites in the liver. This observation, together with sim-ilar findings in other infectious disease models, led to an increasingawareness of organ-specific compartmentalization within the im-mune system following pathogen challenge (8).The infection of genetically susceptible mice with L. donovani

results in parasite growth in the liver, spleen, lymph nodes, andbone marrow (BM) (9–11). Although a chronic infection is es-tablished in the latter three tissue sites, a CD4+ T cell–dependentimmune response develops in the liver, resulting in the formationof proinflammatory granulomas around infected Kupffer cellsassociated with control of infection, although sterilizing immunityis rarely, if ever, observed (12–14). Remarkably, despite thepresence of high parasite burdens in other infected tissues, theliver remains immune to reinfection by mechanisms dependent onCD4+ T cells (15). However, the requirements for the generation

*QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4006, Aus-tralia; †Institute for Glycomics, Griffith University, Gold Coast, Queensland 4215,Australia; ‡Institute for Molecular Biology, University of Queensland, St. Lucia,Queensland 4072, Australia; xRoyal College of Surgeons in Ireland, Dublin D2,Ireland; {London School of Hygiene and Tropical Medicine, London WC1E 7HT,United Kingdom; ‖School of Biomedical Sciences, Queensland University of Tech-nology, Brisbane, Queensland 4001, Australia; #School of Medicine, University ofQueensland, Herston, Queensland 4006, Australia; and **Department of Biology,Hull York Medical School, York University, York YO10 5DD, United Kingdom

Received for publication March 21, 2013. Accepted for publication February 13,2014.

1P.T.B. and A.C.S. contributed equally to this work.

This work was supported by grants from the Australian National Health and MedicalResearch Council and the Australia–India Strategic Research Fund.

Address correspondence and reprint requests to Dr. Christian R. Engwerda, QIMRBerghofer Medical Research Institute, 300 Herston Road, Herston, QLD 4006, Aus-tralia. E-mail address: Christian.Engwerda@qimr.edu.au

Abbreviations used in this article: BM, bone marrow; cDC, conventional dendriticcell; DC, dendritic cell; DT, diphtheria toxin; p.i., postinfection; QIMR, QueenslandInstitute of Medical Research; SLT, secondary lymphoid tissue; VL, visceral leish-maniasis.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300768

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

and maintenance of hepatic CD4+ T cell–mediated immunityduring experimental VL remain poorly understood. Therefore, weinvestigated the anatomical and migratory requirements for thegeneration of CD4+ T cell responses and establishment of im-munity in the liver following L. donovani infection of C57BL/6mice.

Materials and MethodsMice

Inbred female C57BL/6 and B6.SJL.Ptprca (B6.CD45.1) mice were pur-chased from the Australian Resource Centre (Canning Vale, WA, Aus-tralia) and maintained under conventional conditions. B6.RAG12/2 (16),B6.LTa2/2 (17), B6.SJL.Ptprca 3 OT II (18), and B6.CD11c-DTR (19)mice were bred and maintained at the Queensland Institute of MedicalResearch (QIMR). All mice used were matched for age and sex and werehoused under specific pathogen–free conditions. Chimeric mice were pre-pared by irradiating mice with two doses of 5.5 Gy and then engraftingwith 2 3 106 fresh B6.SJL.Ptprca BM cells i.v. via the lateral tail vein.Mice were maintained on antibiotics for 2 wk after engraftment and

infected with L. donovani 8–12 wk after receiving BM, as previously de-scribed (20). In some experiments, mice underwent a splenectomy proce-dure, whereby they were injected with buprenorphine (0.1 mg/kg s.c.) 30min before surgery and then anesthetized with 5% (v/v) isoflurane in 100%(v/v) oxygen. Once unconscious, mice were placed on a heat mat, and an-esthesia was maintained by isoflurane (0.5–1.5% [v/v]) delivered by facemask. Fur was removed using clippers, and skin was sterilized by swabbingwith chlorhexidine in 70% (v/v) ethanol. A 7-mm left paralumbar incisionwas made with a scalpel parallel to the last rib, and the three layers of ab-dominal muscle were split by blunt dissection to expose the spleen. Thespleen was exteriorized and removed using a cauterizing pen. The abdominalmuscles were closed and sutured in one layer, followed by closing of the skinand suturing. Surgical control mice underwent the same procedure, exceptthat the spleen was not removed. Mice were allowed to regain consciousnessin a warm cage and rested for 3–4 wk prior to infection. Depletion of DCs,using diphtheria toxin (DT), was carried out on B6.CD11c-DTR BM chi-meric mice, as previously described (21).

All animal procedures were approved and monitored by the QIMRAnimal Ethics Committee. This work was conducted under QIMR animalethics approval number A02-634M, in accordance with the “AustralianCode of Practice for the Care and Use of Animals for Scientific Purposes”(Australian National Health and Medical Research Council).

FIGURE 1. Ag-specific CD4+ T cell activation is

first observed in the spleen following L. donovani in-

fection. C57BL/6 mice received 2 3 106 CFSE-labeled

OT II cells 2 h prior to infection with OVA-transgenic

L. donovani. At the times indicated, spleen, liver, lymph

nodes (celiac, portal, and mesenteric), and BM were

removed, and CFSE dilution of OT II was analyzed by

FACS. The gating strategy used is shown (top panels).

Graphs show CFSE staining of individual animals in

each group that have been overlaid. The arrow indicates

the first detected proliferating OT II cells on day 2 in

the spleen. CFSE-labeled OT II cells in control mice

5 d p.i. with wild-type (LV9) L. donovani (i.e., no non-

specific CFSE dilution) (far right panels). Bar graphs

show percentage and number of proliferating OT II cells.

Each bar represents the mean 6 SEM. Data are rep-

resentative of two independent experiments (n = 4

mice/group).

3710 HEPATIC IMMUNITY IN EXPERIMENTAL VL

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

Parasites and infections

L. donovani (LV9) and OVA-transgenic LV9 (PINK LV9) (22) weremaintained by passage in B6.RAG12/2 mice, and amastigotes were iso-lated from the spleens of chronically infected mice. Mice were infected byinjecting 2 3 107 amastigotes i.v. via the lateral tail vein, killed at thetimes indicated by CO2 asphyxiation, and bled via cardiac puncture.Spleens and perfused livers were removed at the times indicated, andparasite burdens were determined from Diff-Quick–stained impressionsmears (Lab Aids, Narrabeen, Australia) and expressed as Leishman–Donovan units (number of amastigotes/1000 host nuclei 3 organ weight[g]) (23). Liver tissue also was preserved in Tissue-Tek O.C.T. compound(Sakura, Torrance, CA). Hepatic, splenic, lymph node, and BM mononu-clear cells were isolated as previously described (24).

Anti-CD4 mAb, anti-CD8b mAb, and FTY720 treatment

Ab-producing hybridomas were grown in 5% (v/v) FCS and RPMI 1640containing 10 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml strepto-mycin. Purified Ab was prepared as previously described (25). Mice weredepleted of CD4+ and CD8+ T cells with anti-CD4 (YTS191.1) and anti-CD8b(53-5.8) mAb, respectively, as previously reported (23). Briefly, mice received0.5 mg mAb via the i.p. route every 3 d at the times indicated. Depletion of

T cells was confirmed at the completion of experiments by assessing T cellnumbers in the spleen by flow cytometry. More than 95% of CD4+ and CD8+

T cells were depleted by Ab treatment. In all experiments, control mice re-ceived the same quantities of the appropriate control rat IgG (Sigma-Aldrich).FTY720 (Cayman Chemicals, Ann Arbor, MI) was diluted in saline andinjected via the i.p. route at 1 mg/kg in 150 ml volume on alternate days.

CD4+ T cell proliferation

To assess Ag-specific T cell proliferation in vivo, mice were infected withOVA-transgenic PINK LV9 (22). Splenic OVA-specific OT II T cells wereisolated and labeled with CFSE, as previously described (26). CFSE-labeled OT II cells (2 3 106) were adoptively transferred into mice 2 hprior to infection with LV9 or PINK LV9. Expansion of CFSE+ cells intissues was monitored by FACS at the indicated times. In all of theseexperiments, control animals were included that received the same numberof CFSE-labeled OT II cells but were infected with wild-type parasites. NoOT II proliferation was observed in these animals.

Assessment of granuloma formation

The maturation of granulomas was scored around infected Kupffer cells inacetone-fixed liver sections, as previously described (23, 24).

FIGURE 2. DCs are critical for

Ag-specific CD4+ T cell activation

following L. donovani infection. Chi-

meric B6.CD11c-DTR mice received

2 3 106 CFSE-labeled OT II cells

2 h prior to infection with OVA-

transgenic L. donovani. Half of the

animals were treated with DT to

deplete DCs, whereas the other half

received saline, as indicated. Four

days later, spleen, liver, and lymph

nodes (celiac, portal, and mesen-

teric) were removed, and CFSE di-

lution of OT II was analyzed by

FACS. Graphs show CFSE staining

of individual animals in each group

that have been overlaid. The gating

strategy to measure proliferating OT

II cells is the same as shown in Fig. 1.

Bar graphs show the percentage and

number of proliferating OT II cells

in control and DT-treated mice. Each

bar represents the mean 6 SEM.

Data are representative of three in-

dependent experiments (n = 4 mice/

group). *p , 0.05.

The Journal of Immunology 3711

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

Flow cytometry

Allophycocyanin-conjugated anti–TCRb-chain (H57-597), anti-CD11c(clone N418), PE-Cy5–conjugated anti-CD4 (GK1.5), FITC-conjugatedanti-CD45.1 (clone A20), anti–TCRb-chain (H57-597), Brilliant Violet421–conjugated anti–IFN-g (clone XMG1.2), allophycocyanin-Cy7–conju-gated anti-NK1.1 (clone PK136), Alexa Fluor 700–conjugated anti-CD8a(clone 53-6.7), Pacific Blue–conjugated anti-CD45R/B220 (clone RA3-6B2),and PerCP-Cy5.5–conjugated anti-CD11b (clone M1/70) were purchasedfrom BioLegend (San Diego, CA) or BD Biosciences (Franklin Lakes, NJ).Dead cells were excluded from the analysis using LIVE/DEAD Fixable AquaStain or LIVE/DEAD Fixable Near I-R Stain (Invitrogen-Molecular Probes,Carlsbad, CA), according to the manufacturer’s instructions. The staining ofcell surface Ags and intracellular cytokine staining were carried out as de-scribed previously (25). FACS was performed on a FACSCanto II orLSRFortessa (BD Biosciences), and data were analyzed using FlowJosoftware (TreeStar, Ashland, OR). Gating strategies used for analysis areshown in Figs. 1 and 4.

Statistical analysis

Statistical differences between groups were determined using the Mann–Whitney U test by GraphPad Prism version 4.03 for Windows (GraphPad, SanDiego, CA); p, 0.05 was considered statistically significant. The distributionof hepatic histological responses was compared using the x2 analysis withMicrosoft Excel software. All data are presented as the mean 6 SE, unlessotherwise stated.

ResultsEarly Ag-specific CD4+ T cell expansion occurs in the spleen

To identify where and when Ag-specific CD4+ T cell activationfirst occurs during experimental VL, we transferred 2 3 106

CFSE-labeled OT II cells into C57BL/6 mice infected with OVA-transgenic L. donovani and measured proliferation in the spleen,lymph nodes, liver, and BM each day for the first 5 d of infection(Fig. 1). The celiac, portal, and mesenteric lymph nodes were re-moved for examination in these experiments, because they drainthe liver (27). Notably, OT II proliferation was detected only in thespleen 2 d postinfection (p.i.) (indicated by arrow in Fig. 1). Thefollowing day, dividing OT II cells were observed in all tissuesexamined. We included the BM in this analysis because it is a siteof L. donovani infection (28, 29) and is potentially involved inT cell priming (30). However, after 5 d of infection, the frequencyof OT II cells in BM was less than 10, 2, and 0.5% of that found inthe liver, lymph nodes, and spleen, respectively, suggesting thatthis tissue site played a limited role, if any, in T cell priming.Together, these data indicate that the first round of Ag-specificCD4+ T cell division occurs in the spleen 2 d p.i., prior to thepresence of dividing cells in other infected tissue sites.

FIGURE 3. Ag-specific CD4+ T cell activation

occurs in the liver following L. donovani infection.

C57BL/6 mice received 2 3 106 CFSE-labeled OT

II cells 2 h prior to infection with OVA-transgenic

L. donovani. Mice received either FTY720 or saline, as

indicated. Three days later, spleen, liver, lymph nodes

(celiac, portal, and mesenteric) and BM were removed,

and CFSE dilution of OT II was analyzed by FACS.

Graphs show CFSE staining of individual animals in

each group that have been overlaid. The gating strategy

to measure proliferating OT II cells is the same as

shown in Fig. 1. CFSE-labeled OT II cells in control

mice p.i. with wild-type (LV9) L. donovani (right

panels). Bar graphs show the percentage and number of

proliferating OT II cells in FTY720-treated and con-

trol-treated mice. Each bar represents the mean 6SEM. Data are representative of two independent experi-

ments (n = 4 mice/group). *p , 0.05.

3712 HEPATIC IMMUNITY IN EXPERIMENTAL VL

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

Conventional DCs are critical for Ag-specific CD4+ T cellactivation following L. donovani infection

Despite the widely recognized importance of DCs for Ag-specificactivation of naive CD4+ T cells (31), this has not been demon-strated in experimental VL. To formally test this, we generatedBM chimeric mice in which expression of the DT receptor wasstrictly confined to hematopoietic CD11chi cells (19). These micewere treated with DT or saline, given 2 3 106 CFSE-labeled OT IIcells, and infected with OVA-transgenic L. donovani. OT II pro-liferation in the spleen, liver, and lymph nodes was measured onday 4 p.i. (Fig. 2). As previously reported (21), DT treatment re-sulted in .90% depletion of conventional DCs (cDCs; CD11chi

MHCIIhi), but not plasmacytoid DCs (CD11cint B220/BST2+), inthe spleen (data not shown). Proliferating OT II cells were foundin all tissues from control mice, whereas no OT II cell prolifera-tion was observed in any tissue of mice depleted of cDCs (Fig. 2).These findings formally demonstrate that cDCs are critical for Ag-specific CD4+ T cell activation following L. donovani infection.

Ag-specific CD4+ T cell activation occurs in the liver of L.donovani–infected mice

To test whether Ag-specific CD4+ T cells divide in the spleenbefore migrating to other tissues, we again transferred OT II cellsinto C57BL/6 mice infected with OVA-transgenic L. donovani,and in addition, treated them with FTY720 to prevent lymphocyteegress from the spleen and all other secondary lymphoid tissues(SLTs) (32, 33). As expected, dividing cells were observed in alltissues from control mice 3 d p.i. (Fig. 3). However, althoughFTY720 treatment had no effect on OT II proliferation in thespleen, it significantly inhibited the appearance of proliferating OTII cells in lymph nodes, suggesting that Ag-specific CD4+ T cellsare not activated in these tissues but, once activated, can trafficthrough them. However, it should be noted that this blockade wasnot complete, and some proliferation of Ag-specific CD4+ T cells

was still observed in lymph nodes of drug-treated mice. In con-trast, FTY720 had minimal impact on OT II division in the liver,indicating that Ag-specific CD4+ T cell activation occurs in thisorgan. Together, these data indicate that Ag-specific CD4+ T cellactivation following L. donovani infection primarily occurs in thespleen and liver, but the spleen appears to play a greater role inthis process, as indicated by a larger number of proliferating, Ag-specific CD4+ T cells in this tissue site.

FTY720 treatment prevents CD4+ T cell–mediated immunityfrom being established in the liver

Our results above indicate that antiparasitic CD4+ T cell responseswere generated in the liver. To examine whether these responseswere capable of mediating efficient antiparasitic immunity, we nextassessed hepatic parasite burdens in mice continuously treated withFTY720 to block T cell trafficking from the spleen and lymphnodes to the liver over the first month of infection (Fig. 4). Asexpected from previous studies (34), depletion of CD4+ T cells overthis time caused increased parasite growth in mice by day 28 p.i.(Fig. 4A). Importantly, mice treated with FTY720 also had elevatedparasite burdens by day 28 p.i. compared with control animals(Fig. 4A), suggesting that parasite-specific CD4+ T cells had totraffic through SLTs and back into the liver to control hepatic in-fection. In support of this, we found reduced numbers of livermononuclear cells (Fig. 4C), total CD4+ T cells (Fig. 4D), and IFN-g–producing CD4+ T cells (Fig. 4B, 4E) at day 28 p.i. in micetreated with FTY720 compared with control animals. AlthoughFTY720 reduced the number of IFN-g–producing CD4+ T cells(Fig. 4E), the frequency of these cells was not different (Fig. 4B).Thus, we cannot exclude the possibility that the drug might reduceCD4+ T cells in the liver without impacting upon the ability toprime antiparasitic CD4+ T cell responses.FTY720 was reported to alter the positioning of DCs within the

spleen (35), as well as reduce splenic DC number and frequency of

FIGURE 4. CD4+ T cell migra-

tion through SLTs is required for

early control of parasite growth in

the liver following L. donovani in-

fection. (A) Infected mice were treated

with anti-CD4 mAb (s), control Ab

(d), FTY720 (O), or saline (:) from

the start of infection, and hepatic

parasite burdens were measured at

indicated times. The gating strategy

to measure hepatic cell populations

is the same as shown in Fig. 1 for

live cells and then as depicted in (B).

Representative plots from control

and FTY720-treated groups at day

28 p.i. Hepatic mononuclear cell

(MNC) number (C), CD4+ T cell

number (D), and IFN-g–producing

CD4+ T cell number (E) also were

measured at day 28 p.i. in FTY720-

treated and saline-treated mice. Each

bar represents the mean 6 SEM. Data

are representative of two independent

experiments (n = 4 mice/group). *p ,0.05, **p , 0.01.

The Journal of Immunology 3713

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

responder T cells in a model of graft-versus-host disease (36).Therefore, a more detailed analysis of the kinetics of CD4+ T cellresponses in the liver and spleen of L. donovani–infected micetreated for 28 d with FTY720 was undertaken (Fig. 5), includingexamination of expression of CD49d and CD11a activationmarkers on CD4+ T cells, because these were reported to markAg-experienced CD4+ T cells (37). We found increased hepaticparasite burden (Fig. 5A) and decreased hepatic mononuclear cell(Fig. 5B) and CD4+ T cell (Fig. 5C) numbers, accompanied bya reduction in the number of Ag-experienced CD4+ T cells(Fig. 5D) in FTY720-treated mice compared with control mice,as well as similar effects in the spleen at day 28 p.i. (Fig. 5, rightpanels). Thus, these data suggest that FTY720 has other effects onCD4+ T cell activation following L. donovani infection not relatedto blocking egress of lymphocytes out of SLTs.

Maintenance of hepatic immunity and control of parasitegrowth in the chronically infected spleen require CD4+ T cells

We next investigated the impact of CD4+ and CD8+ T cell de-pletion on the maintenance of antiparasitic immunity at day 56p.i., after parasite-specific CD4+ T cells had been generated and

controlled infection in the liver but not the spleen. Depletion ofCD4+ T cells in mice between days 56 and 70 p.i. caused in-creased parasite growth in the liver and spleen (Fig. 6A). Thisconfirmed a key role for these cells in maintaining immunity in theliver (15) and showed that they mediate control over parasitegrowth in the spleen during chronic infection. In contrast, deple-tion of CD8+ T cells at this time had no impact on parasite bur-dens. We also delayed CD4+ T cell depletion until a later time(days 90–104 p.i.) to establish whether protective CD4+ T cell–mediated immunity diminished. However, we again found thatdepletion of CD4+ T cells over this time caused increased parasitegrowth in the liver and spleen (Fig. 6B), confirming a critical rolefor this cell population in the maintenance of hepatic immunityand control of parasite growth in the chronically infected spleen.

Hepatic immunity can be generated in the complete absence ofSLT following L. donovani infection

Finally, as an alternative way to test whether antiparasitic immunityin the liver required any SLT involvement, we generated micelacking a spleen and/or lymph nodes by engrafting wild-type BMinto lethally irradiated B6.LTa2/2 mice, which lack all lymph

FIGURE 5. FTY720 impairs con-

trol of parasite growth and reduces cell

numbers in the spleen and liver of

L. donovani–infected mice. (A) Infec-

ted mice were treated with FTY720

(O) or saline (:) over the course of

infection, and parasite burdens in

the liver and spleen were measured

at the indicated times. The gating

strategy to measure hepatic cell pop-

ulations was the same as shown in

Fig. 4. Mononuclear cell (MNC)

number (B), CD4+ T cell number

(C), and Ag-experienced (CD11b+,

CD49d+) CD4+ T cells (D) were also

measured at the indicated times. Each

data point represents the mean 6SEM (n = 5 mice/group). *p , 0.05,

**p , 0.01, ***p , 0.001.

3714 HEPATIC IMMUNITY IN EXPERIMENTAL VL

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

nodes (17), and then splenectomizing or subjecting them to amock surgical procedure. Mice lacking a spleen, lymph nodes,or both were able to control L. donovani infection in the liver withsimilar kinetics to control animals (Fig. 7A). Furthermore, nodifferences in hepatic granuloma development, a critical CD4+

T cell–dependent, antiparasitic mechanism (34), were observed byday 28 p.i. (Fig. 7B). Importantly, when mice were reinfected atday 56 p.i., parasite growth was controlled rapidly in all groups(Fig. 7A, left panel) relative to age-matched, naive C57BL/6 mice(Fig. 7A, right panel). In addition, there were increased numbersof liver mononuclear cells, total CD4+ T cells, and IFN-g–pro-ducing CD4+ T cells (Fig. 7C–E) in all groups after both primaryand secondary infection. Together, these data clearly indicate thatantiparasitic immunity in the liver can be generated and main-tained in the absence of SLTs.

DiscussionL. donovani infects and resides in tissue macrophages of thespleen, liver, lymph nodes, and BM. However, the extent of dis-ease in these different tissue sites varies considerably amonghuman VL patients (38). C57BL/6 mice experimentally infectedwith this parasite display one pattern of this disease profile,whereby the spleen and BM harbor relatively high parasite bur-dens for life, associated with considerable tissue pathology,whereas the liver is a site of resolving infection that becomesimmune to reinfection (7, 8, 34). As such, this experimental modelprovides the opportunity to study the establishment of immunity inliver, as well as factors that promote chronic infection and diseasepathogenesis in the spleen in the same infected animal. Given theimportance of secondary lymphoid structure for the activation ofCD4+ T cells (39), as well as the maintenance of CD4+ T cellmemory by enabling appropriate lymphocyte movement (40), weexamined whether protective CD4+ T cell immunity could developand be maintained in the liver. Our findings indicate that Ag-specific CD4+ T cell responses can be generated and maintained

in the liver of genetically susceptible mice following L. donovaniinfection. Furthermore, we also found that CD4+ T cells playeda critical role in controlling parasite growth in chronically infectedspleens.Previous reports indicated that CD4+ T cells from VL patients

were refractory to Ag stimulation, as indicated by a lack of re-sponse in the leishmanin skin test and a failure of PBMCs toproliferate and produce IFN-g in response to parasite Ags (41).However, more recent work showed that CD4+ T cells from VLpatients can respond to parasite Ag in whole-blood assays, sug-gesting that sample processing may have contributed to earlierresults (42–44). Our results showing that CD4+ T cells were im-portant for controlling parasite burden in chronically infectedspleens of L. donovani–infected mice (Fig. 6) support the idea thatCD4+ T cells in chronically infected tissues are not refractory toAg stimulation and potentially can be targeted for therapeuticadvantage. IL-10 has been identified as an important suppressor ofparasite-specific CD4+ T cell responses in VL patients (44, 45);therefore, transient blockade of this cytokine or similar immune-suppressive molecules may be one approach to harnessing theantiparasitic potential of CD4+ T cells in infected individuals.Previous work with alymphoplastic mice suggested that CD4+

and CD8+ T cell responses can be generated in the liver in theabsence of SLTs (46). Similarly, lymphoid tissues in the lung(BALT) were reported to generate primary T cell responsesagainst influenza (47), supporting the idea that naive T cells canbe activated in nonlymphoid tissues. However, in both of thesestudies, the importance of nonlymphoid T cell activation in tissue-intact animals was not fully addressed. Furthermore, the possi-bility that T cell activation might occur in the BM, as reported byother investigators (30), was not excluded. Nevertheless, elegantstudies using liver transplantation in mice with SLTs showed thatAg-specific CD8+ T cells could be activated in recipient mice,even though the recipients’ MHC class I molecules were unable topresent the antigenic peptide (48), thus providing strong evidencefor the activation of naive CD8+ T cells in nonlymphoid tissues.Our results indicate that there were 10–100–fold more Ag-

specific CD4+ T cells in the spleen compared with the liver inthe first few days of L. donovani infection (Fig. 1), suggesting thatthis tissue is normally the major site for early CD4+ T cell acti-vation in this model. However, the liver is a site where relativelylarge numbers of NKT cells reside (49), and we showed previ-ously that these cells expand and become activated followingL. donovani infection, although they only play a minor role in con-trolling parasite growth (24). Our studies in mice lacking lymphnodes and spleen indicate that activation of conventional, Ag-specific CD4+ T cells also can occur in the liver during ex-perimental VL. In addition, in FTY720-treated animals, whenAg-specific CD4+ T cell trafficking to lymph nodes had beenblocked, albeit not completely (Fig. 3), Ag-specific CD4+ T cellactivation was able to occur in both the spleen and liver, con-firming that Ag-specific CD4+ T cell activation could occur in theliver. The finding that hepatic immunity could be generated inmice lacking a spleen, lymph nodes, or both (Fig. 7) furthersupports this conclusion and also indicates that SLTs were notrequired for the establishment and maintenance of this immunity.The ability of conventional, antiparasitic CD4+ T cells activated

in the liver to maintain residency in this organ was difficult toassess because of the potential effects of long-term FTY720treatment not relating to blockade of lymphocyte egress fromSLTs (Figs. 4, 5). T cells move through the liver via sinusoids, andintravital two-photon imaging studies on Ag-specific CD8+ T cellmovements through the liver in L. donovani–infected mice showthat these cells leave the sinusoids and enter granulomas formed

FIGURE 6. CD4+ T cells are critical for control of parasite growth in

mice with established L. donovani infection. Mice with established in-

fection were treated with anti-CD4 mAb, anti-CD8b mAb, or control Ab

between days 57 and 70 p.i. (A), or with anti-CD4 mAb or control Ab

between days 70 and 90 p.i. (B), and parasite burden in the spleen and liver

was measured. Each bar represents the mean 6 SEM. Data are represen-

tative of two or three independent experiments at each time point (n = 4–5

mice/group). *p , 0.05, **p , 0.01, ***p , 0.001.

The Journal of Immunology 3715

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

around infected Kupffer cells, where they appear to sample cellsurfaces before exiting the granuloma structure back into thesinusoids (50). Whether Ag-specific CD4+ T cells exhibit a similarpattern of cell movement in this model is not known. However,when hepatic immunity has been established, they are critical forcontrolling persisting parasites in this tissue site. Whether thesecells are true liver-resident cells has not been formally established;however, if this were the case, one possible location for them wouldbe in the hepatic granulomas that remain intact long after parasitegrowth has been controlled in this tissue ($180 d; C.R. Engwerda,unpublished observations). It should be emphasized that sterilizingimmunity is not established in this model, and that at least some ofthese remaining hepatic granulomas are likely to be sites for lownumbers of persisting parasites, as borne out in recent computa-tional modeling of granuloma diversity (51). Such granulomas mayprovide the location for sustained local Ag presentation to parasite-specific CD4+ T cells. Thus, our findings suggest the existence ofa tissue-resident CD4+ T cell population responsible for maintainingimmunity in the liver. Importantly, a large-scale genome-wide as-sociation study (52) on populations from India and Brazil recentlyidentified HLA class II region polymorphisms as important geneticrisk factors for VL, indicating that CD4+ T cells are likely to playkey roles in determining the outcome of VL in humans.

As mentioned above, when FTY720 was administered earlyduring infection (days 0–28 p.i.), there appeared to be drug effectsnot related to blocking lymphocyte egress out of SLTs, becauseantiparasitic immunity, including numbers of Ag-experienced CD4+

T cells, was diminished in the spleen, as well as the liver (Fig. 5).If FTY720 was simply preventing lymphocyte egress from SLTs,then one might predict an accumulation of lymphocytes in thesetissue sites. Previous work (36) in a model of graft-versus-hostdisease reported similar findings, which may be related to theeffects of FTY720 on DCs’ ability to move in the spleen andposition themselves for efficient CD4+ T cell activation (35). Ourresults indicate a similar effect; however, because the drug did notinterfere with Ag-specific CD4+ T cell proliferation in the first 3 dof infection (Fig. 3), these effects appear to impact upon DCsrecruited to tissues p.i. In experiments involving FTY720 treat-ment during established infection, we also observed decreasedmononuclear cell numbers in the liver and spleen (data not shown),consistent with previous reports showing that long-term drugtreatment results in decreased lymphocyte numbers in the body(53). Thus, we were unable to assess the requirements for tissuemigration of CD4+ T cells on the maintenance of antiparasiticimmunity in infected organs. However, it was clear from ourfindings in alymphoplastic mice (Fig. 7) that potent hepatic anti-

FIGURE 7. Control of L. dono-

vani infection in the liver can be

achieved in the absence of the spleen

and lymph nodes. Chimeric mice

were generated by lethally irradiat-

ing C57BL/6 or B6.LTa2/2 mice

and then engrafting with congenic

(CD45.1) BM. Half of the C57BL/6

(n) and B6.LTa2/2 (P) recipients

underwent a mock surgical procedure,

whereas the other half of the C57BL/6

(:) and B6.LTa2/2 ()) recipients

were splenectomized (SPx). (A) Mice

were infected with L. donovani, and

liver parasite burden was measured

over the course of the infection (left

panel). The arrow indicates when

animals were reinfected with 2 3107 L. donovani. Bar graph shows

day-14 p.i. liver parasite burdens in

naive, age-matched C57BL/6 mice

infected at the same time (right panel).

(B) Liver granuloma formation was

assessed by counting the numbers

of infected Kupffer cells without

inflammatory foci (white bars) or

with immature (gray bars) or mature

(black bars) granulomas at day 28

p.i. The gating strategy to measure

hepatic cell populations was the same

as shown in Fig. 4. Hepatic mono-

nuclear cell (MNC) number (C), CD4+

T cell number (D), and IFN-g–pro-

ducing CD4+ T cell number (E) were

measured over the course of infection.

The arrow indicates when animals

were reinfected, as in (A). Data are

representative of two or three inde-

pendent experiments (n = 4–5 mice/

group at each time point).

3716 HEPATIC IMMUNITY IN EXPERIMENTAL VL

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

parasitic CD4+ T cell responses could be generated and main-tained in the absence of SLTs.One unexpected finding from our studies was the amount of time

taken before CD4+ T cell responses were able to mediate controlof parasite growth in the liver. The impact of CD4+ T cell de-pletion was not observed until after day 14 p.i. (Fig. 4A), whereasprevious studies in B6.RAG12/2 mice indicated a clear defect inthe control of parasite growth by this time point (26, 54, 55).Hence, either immune regulatory pathways are initiated soon p.i.that can suppress antiparasitic CD4+ T cell development or someearly antiparasitic activity can be generated in the absence ofCD4+ T cells. In support of the former possibility, we observedthat Ag-specific (OT II) CD4+ T cell numbers increased for 3 dfollowing infection in the spleen and 4 d in the liver and lymphnodes and then stopped (Fig. 1). The reason for this halt in cellexpansion is not clear, but likely reflects a mechanism of immunemodulation contributing to inefficient control of parasite growth inthe liver and the establishment of a chronic infection in the spleenand BM. Regardless, CD4+ T cells ultimately are able to controlparasite growth in the liver (Fig. 4) and are required for continuedsurveillance in this tissue site (Fig. 6) (15), as well as containingparasite numbers in the spleen during the chronic phase of in-fection (Fig. 6).In conclusion, we demonstrate that DCs are critical for CD4+

T cell activation following L. donovani infection and that anti-parasitic CD4+ T cell responses can be generated and maintainimmunity in the liver. If we can better understand how theseprotective CD4+ T cell responses are generated, we may be able totarget vaccines or therapies for their more efficient development.Our finding that CD4+ T cells play an active role in controllingparasite growth in the spleen during chronic infection also indi-cates that they can be targeted to better eliminate parasites fromthis tissue. Thus, our work identifies new approaches to try andcontrol an important parasitic disease of humans.

AcknowledgmentsWe thank Ian Shiels, Craig Dickfos, and Dianne Carrie for performing an-

imal surgery. We also thank Paula Hall and Grace Chojnowski for assistance

with flow cytometry and staff in the QIMR Berghofer animal facility for

animal husbandry.

DisclosuresThe authors have no financial conflicts of interest.

References1. Kraal, G. 1992. Cells in the marginal zone of the spleen. Int. Rev. Cytol. 132: 31–

74.2. Mebius, R. E., and G. Kraal. 2005. Structure and function of the spleen. Nat. Rev.

Immunol. 5: 606–616.3. Boone, K. E., and D. A. Watters. 1995. The incidence of malaria after sple-

nectomy in Papua New Guinea. BMJ 311: 1273.4. Davidson, R. N., and R. A. Wall. 2001. Prevention and management of infections

in patients without a spleen. Clin. Microbiol. Infect. 7: 657–660.5. Kruetzmann, S., M. M. Rosado, H. Weber, U. Germing, O. Tournilhac,

H. H. Peter, R. Berner, A. Peters, T. Boehm, A. Plebani, et al. 2003. Humanimmunoglobulin M memory B cells controlling Streptococcus pneumoniaeinfections are generated in the spleen. J. Exp. Med. 197: 939–945.

6. Kaye, P. M., M. Svensson, M. Ato, A. Maroof, R. Polley, S. Stager, S. Zubairi,and C. R. Engwerda. 2004. The immunopathology of experimental visceralleishmaniasis. Immunol. Rev. 201: 239–253.

7. Stanley, A. C., and C. R. Engwerda. 2007. Balancing immunity and pathology invisceral leishmaniasis. Immunol. Cell Biol. 85: 138–147.

8. Engwerda, C. R., and P. M. Kaye. 2000. Organ-specific immune responses as-sociated with infectious disease. Immunol. Today 21: 73–78.

9. Smelt, S. C., C. R. Engwerda, M. McCrossen, and P. M. Kaye. 1997. Destructionof follicular dendritic cells during chronic visceral leishmaniasis. J. Immunol.158: 3813–3821.

10. Bryceson, A. 1996. Tropical medicine for the 21st century. Tropical medicineshould be concerned with medical problems endemic to the tropics. BMJ 312: 247.

11. Wilson, M. E., M. Sandor, A. M. Blum, B. M. Young, A. Metwali, D. Elliott,R. G. Lynch, and J. V. Weinstock. 1996. Local suppression of IFN-gamma inhepatic granulomas correlates with tissue-specific replication of Leishmaniachagasi. J. Immunol. 156: 2231–2239.

12. Crocker, P. R., J. M. Blackwell, and D. J. Bradley. 1984. Expression of thenatural resistance gene Lsh in resident liver macrophages. Infect. Immun. 43:1033–1040.

13. McElrath, M. J., H. W. Murray, and Z. A. Cohn. 1988. The dynamics of gran-uloma formation in experimental visceral leishmaniasis. J. Exp. Med. 167: 1927–1937.

14. Murray, H. W. 2001. Tissue granuloma structure-function in experimental vis-ceral leishmaniasis. Int. J. Exp. Pathol. 82: 249–267.

15. Murray, H. W., J. Hariprashad, and R. E. Fichtl. 1996. Models of relapse ofexperimental visceral leishmaniasis. J. Infect. Dis. 173: 1041–1043.

16. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, andV. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B andT lymphocytes. Cell 68: 869–877.

17. Sean Riminton, D., H. Korner, D. H. Strickland, F. A. Lemckert, J. D. Pollard,and J. D. Sedgwick. 1998. Challenging cytokine redundancy: inflammatory cellmovement and clinical course of experimental autoimmune encephalomyelitisare normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient,mice. J. Exp. Med. 187: 1517–1528.

18. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCRexpression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol.Cell Biol. 76: 34–40.

19. Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu,S. Vuthoori, K. Ko, F. Zavala, et al. 2002. In vivo depletion of CD11c+ dendriticcells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.Immunity 17: 211–220.

20. Haque, A., S. E. Best, A. Ammerdorffer, L. Desbarrieres, M. M. de Oca,F. H. Amante, F. de Labastida Rivera, P. Hertzog, G. M. Boyle, G. R. Hill, andC. R. Engwerda. 2011. Type I interferons suppress CD4+ T-cell-dependentparasite control during blood-stage Plasmodium infection. Eur. J. Immunol.41: 2688–2698.

21. deWalick, S., F. H. Amante, K. A. McSweeney, L. M. Randall, A. C. Stanley,A. Haque, R. D. Kuns, K. P. MacDonald, G. R. Hill, and C. R. Engwerda. 2007.Cutting edge: conventional dendritic cells are the critical APC required for theinduction of experimental cerebral malaria. J. Immunol. 178: 6033–6037.

22. Polley, R., S. Stager, S. Prickett, A. Maroof, S. Zubairi, D. F. Smith, andP. M. Kaye. 2006. Adoptive immunotherapy against experimental visceralleishmaniasis with CD8+ T cells requires the presence of cognate antigen. Infect.Immun. 74: 773–776.

23. Haque, A., A. C. Stanley, F. H. Amante, Fde. L. Rivera, Y. Zhou, R. D. Kuns,V. Yardley, S. Sakaguchi, G. R. Hill, and C. R. Engwerda. 2010. Therapeuticglucocorticoid-induced TNF receptor-mediated amplification of CD4+ T cellresponses enhances antiparasitic immunity. J. Immunol. 184: 2583–2592.

24. Stanley, A. C., Y. Zhou, F. H. Amante, L. M. Randall, A. Haque, D. G. Pellicci,G. R. Hill, M. J. Smyth, D. I. Godfrey, and C. R. Engwerda. 2008. Activation ofinvariant NKT cells exacerbates experimental visceral leishmaniasis. PLoSPathog. 4: e1000028.

25. Stanley, A. C., J. E. Dalton, S. H. Rossotti, K. P. MacDonald, Y. Zhou, F. Rivera,W. A. Schroder, A. Maroof, G. R. Hill, P. M. Kaye, and C. R. Engwerda. 2008.VCAM-1 and VLA-4 modulate dendritic cell IL-12p40 production in experi-mental visceral leishmaniasis. PLoS Pathog. 4: e1000158.

26. Stanley, A. C., F. de Labastida Rivera, A. Haque, M. Sheel, Y. Zhou,F. H. Amante, P. T. Bunn, L. M. Randall, K. Pfeffer, S. Scheu, et al. 2011.Critical roles for LIGHT and its receptors in generating T cell-mediated im-munity during Leishmania donovani infection. PLoS Pathog. 7: e1002279.

27. Barbier, L., S. S. Tay, C. McGuffog, J. A. Triccas, G. W. McCaughan,D. G. Bowen, and P. Bertolino. 2012. Two lymph nodes draining the mouse liverare the preferential site of DC migration and T cell activation. J. Hepatol. 57:352–358.

28. Cotterell, S. E., C. R. Engwerda, and P. M. Kaye. 2000. Enhanced hematopoieticactivity accompanies parasite expansion in the spleen and bone marrow of miceinfected with Leishmania donovani. Infect. Immun. 68: 1840–1848.

29. Cotterell, S. E., C. R. Engwerda, and P. M. Kaye. 2000. Leishmania donovaniinfection of bone marrow stromal macrophages selectively enhances myelo-poiesis, by a mechanism involving GM-CSF and TNF-alpha. Blood 95: 1642–1651.

30. Feuerer, M., P. Beckhove, N. Garbi, Y. Mahnke, A. Limmer, M. Hommel,G. J. Hammerling, B. Kyewski, A. Hamann, V. Umansky, and V. Schirrmacher.2003. Bone marrow as a priming site for T-cell responses to blood-borne antigen.Nat. Med. 9: 1151–1157.

31. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu,B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu.Rev. Immunol. 18: 767–811.

32. Brinkmann, V., M. D. Davis, C. E. Heise, R. Albert, S. Cottens, R. Hof, C. Bruns,E. Prieschl, T. Baumruker, P. Hiestand, et al. 2002. The immune modulatorFTY720 targets sphingosine 1-phosphate receptors. J. Biol. Chem. 277: 21453–21457.

33. Mandala, S., R. Hajdu, J. Bergstrom, E. Quackenbush, J. Xie, J. Milligan,R. Thornton, G. J. Shei, D. Card, C. Keohane, et al. 2002. Alteration oflymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science296: 346–349.

34. Murray, H. W., K. E. Squires, C. D. Miralles, M. Y. Stoeckle, A. M. Granger,A. Granelli-Piperno, and C. Bogdan. 1992. Acquired resistance and granuloma

The Journal of Immunology 3717

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from

formation in experimental visceral leishmaniasis. Differential T cell andlymphokine roles in initial versus established immunity. J. Immunol. 148:1858–1863.

35. Czeloth, N., A. Schippers, N. Wagner, W. M€uller, B. K€uster, G. Bernhardt, andR. Forster. 2007. Sphingosine-1 phosphate signaling regulates positioning ofdendritic cells within the spleen. J. Immunol. 179: 5855–5863.

36. Taylor, P. A., M. J. Ehrhardt, C. J. Lees, J. Tolar, B. J. Weigel, A. Panoskaltsis-Mortari, J. S. Serody, V. Brinkmann, and B. R. Blazar. 2007. Insights into themechanism of FTY720 and compatibility with regulatory T cells for the inhi-bition of graft-versus-host disease (GVHD). Blood 110: 3480–3488.

37. Butler, N. S., J. Moebius, L. L. Pewe, B. Traore, O. K. Doumbo, L. T. Tygrett,T. J. Waldschmidt, P. D. Crompton, and J. T. Harty. 2011. Therapeutic blockadeof PD-L1 and LAG-3 rapidly clears established blood-stage Plasmodium in-fection. Nat. Immunol. 13: 188–195.

38. Bryceson, A. D. M. 1996. Leishmaniasis. In Manson’s Tropical Diseases, 20thEd. G. C. Cook, ed. W. B. Saunders, London, p. 1213–1245.

39. Takada, K., and S. C. Jameson. 2009. Naive T cell homeostasis: from awarenessof space to a sense of place. Nat. Rev. Immunol. 9: 823–832.

40. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Twosubsets of memory T lymphocytes with distinct homing potentials and effectorfunctions. Nature 401: 708–712.

41. Sacks, D. L., S. L. Lal, S. N. Shrivastava, J. Blackwell, and F. A. Neva. 1987. Ananalysis of T cell responsiveness in Indian kala-azar. J. Immunol. 138: 908–913.

42. Ansari, N. A., R. Kumar, S. Gautam, S. Nylen, O. P. Singh, S. Sundar, andD. Sacks. 2011. IL-27 and IL-21 are associated with T cell IL-10 responses inhuman visceral leishmaniasis. J. Immunol. 186: 3977–3985.

43. Gidwani, K., A. Picado, S. Rijal, S. P. Singh, L. Roy, V. Volfova, E. W. Andersen,S. Uranw, B. Ostyn, M. Sudarshan, et al. 2011. Serological markers of sand flyexposure to evaluate insecticidal nets against visceral leishmaniasis in India andNepal: a cluster-randomized trial. PLoS Negl. Trop. Dis. 5: e1296.

44. Singh, O. P., K. Gidwani, R. Kumar, S. Nylen, S. L. Jones, M. Boelaert,D. Sacks, and S. Sundar. 2012. Reassessment of immune correlates in humanvisceral leishmaniasis as defined by cytokine release in whole blood. Clin.Vaccine Immunol. 19: 961–966.

45. Gautam, S., R. Kumar, R. Maurya, S. Nylen, N. Ansari, M. Rai, S. Sundar, andD. Sacks. 2011. IL-10 neutralization promotes parasite clearance in splenic aspiratecells from patients with visceral leishmaniasis. J. Infect. Dis. 204: 1134–1137.

46. Greter, M., J. Hofmann, and B. Becher. 2009. Neo-lymphoid aggregates in theadult liver can initiate potent cell-mediated immunity. PLoS Biol. 7: e1000109.

47. Moyron-Quiroz, J. E., J. Rangel-Moreno, K. Kusser, L. Hartson, F. Sprague,S. Goodrich, D. L. Woodland, F. E. Lund, and T. D. Randall. 2004. Role ofinducible bronchus associated lymphoid tissue (iBALT) in respiratory immunity.Nat. Med. 10: 927–934.

48. Klein, I., and I. N. Crispe. 2006. Complete differentiation of CD8+ T cells ac-tivated locally within the transplanted liver. J. Exp. Med. 203: 437–447.

49. Godfrey, D. I., H. R. MacDonald, M. Kronenberg, M. J. Smyth, and L. Van Kaer.2004. NKT cells: what’s in a name? Nat. Rev. Immunol. 4: 231–237.

50. Beattie, L., A. Peltan, A. Maroof, A. Kirby, N. Brown, M. Coles, D. F. Smith,and P. M. Kaye. 2010. Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentationto antigen-specific CD8 T cells. PLoS Pathog. 6: e1000805.

51. Albergante, L., J. Timmis, L. Beattie, and P. M. Kaye. 2013. A Petri net model ofgranulomatous inflammation: implications for IL-10 mediated control ofLeishmania donovani infection. PLOS Comput. Biol. 9: e1003334.

52. Fakiola, M., A. Strange, H. J. Cordell, E. N. Miller, M. Pirinen, Z. Su, A. Mishra,S. Mehrotra, G. R. Monteiro, G. Band, et al; LeishGEN Consortium; WellcomeTrust Case Control Consortium 2. 2013. Common variants in the HLA-DRB1-HLA-DQA1 HLA class II region are associated with susceptibility to visceralleishmaniasis. Nat. Genet. 45: 208–213.

53. Morris, M. A., D. R. Gibb, F. Picard, V. Brinkmann, M. Straume, and K. Ley.2005. Transient T cell accumulation in lymph nodes and sustained lymphopeniain mice treated with FTY720. Eur. J. Immunol. 35: 3570–3580.

54. Engwerda, C. R., M. Ato, S. Stager, C. E. Alexander, A. C. Stanley, andP. M. Kaye. 2004. Distinct roles for lymphotoxin-alpha and tumor necrosis factorin the control of Leishmania donovani infection. Am. J. Pathol. 165: 2123–2133.

55. Alexander, C. E., P. M. Kaye, and C. R. Engwerda. 2001. CD95 is required forthe early control of parasite burden in the liver of Leishmania donovani-infectedmice. Eur. J. Immunol. 31: 1199–1210.

3718 HEPATIC IMMUNITY IN EXPERIMENTAL VL

at Queensland Institute of M

edical Research on July 1, 2014

http://ww

w.jim

munol.org/

Dow

nloaded from