Cutaneous Infection with major Mediates Heterologous Protection against Visceral Infection with Leishmania infantum

This information is current as Audrey Romano, Nicole A. Doria, Jonatan Mendez, David of October 2, 2021. L. Sacks and Nathan C. Peters J Immunol 2015; 195:3816-3827; Prepublished online 14 September 2015; doi: 10.4049/jimmunol.1500752 http://www.jimmunol.org/content/195/8/3816 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2015/09/13/jimmunol.150075 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 73 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/195/8/3816.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cutaneous Infection with Mediates Heterologous Protection against Visceral Infection with Leishmania infantum

Audrey Romano,* Nicole A. Doria,* Jonatan Mendez,* David L. Sacks,* and Nathan C. Peters†

Visceral (VL) is a fatal disease of the internal organs caused by the eukaryotic parasite Leishmania. Control of VL would best be achieved through vaccination. However, this has proven to be difficult partly because the correlates of protective immunity are not fully understood. In contrast, protective immunity against nonfatal (CL) is well defined and mediated by rapidly recruited, IFN-g–producing Ly6C+CD4+ T cells at the dermal challenge site. Protection against CL is best achieved by prior infection or live vaccination with Leishmania major, termed leishmanization. A long-standing question is whether prior CL or leishmanization can protect against VL. Employing an intradermal challenge model in mice, we report that Downloaded from cutaneous infection with Leishmania major provides heterologous protection against visceral infection with Leishmania infantum. Protection was associated with a robust CD4+ T cell response at the dermal challenge site and in the viscera. In vivo labeling of circulating cells revealed that increased frequencies of IFN-g+CD4+ T cells at sites of infection are due to recruitment or retention of cells in the tissue, rather than increased numbers of cells trapped in the vasculature. Shortly after challenge, IFN-g–producing cells were highly enriched for Ly6C+T-bet+ cells in the viscera. Surprisingly, this heterologous immunity was superior to homol- ogous immunity mediated by prior infection with L. infantum. Our observations demonstrate a common mechanism of protection http://www.jimmunol.org/ against different clinical forms of leishmaniasis. The efficacy of leishmanization against VL may warrant the introduction of the practice in VL endemic areas or during outbreaks of disease. The Journal of Immunology, 2015, 195: 3816–3827.

he leishmaniases consist of a broad range of cutaneous, reached epidemic proportions and are thought to be a major mucocutaneous, and visceral diseases caused by different reservoir of human disease (7). T strains of the eukarytotic parasite Leishmania. Leish- No vaccine is currently available for any form of leishmaniasis mania, which is transmitted to the human host by the bite of in people. However, a deliberate, single needle inoculation of a sand fly vector, is an obligate intracellular pathogen that es- infectious Leishmania major into the skin without the disease- by guest on October 2, 2021 tablishes chronic infection within phagocytes. Visceral forms of exacerbating factors coegested during natural sand fly transmis- leishmaniasis are fatal when left untreated, and although drugs are sion, termed leishmanization, provides complete and long-lasting available they are expensive, highly toxic, and drug resistance is homologous protection against sand fly–transmitted cutaneous common (1, 2). caused by Leishmania disease and has been used extensively as a live vaccine in humans donovani is endemic in northern India and East Africa, whereas (8–12). Despite its efficacy and the convenience of a single ad- Leishmania infantum, also known as Leishmania chagasi, is the ministration, leishmanization has largely been abandoned because causative agent of visceral leishmaniasis (VL) in South America of rare adverse reactions at the site of inoculation (13, 14); fur- and the Mediterranean basin, which has experienced several recent thermore, the chronic nature of the infection raises concerns outbreaks (3–6). In some areas, L. infantum infection of dogs has should leishmanized individuals become immunocompromised, although there are no reports of reactivation or dissemination of

*Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Dis- L. major in leishmanized individuals. A more justifiable use of eases, National Institutes of Health, Bethesda, MD 20892; and †Snyder Institute for leishmanization would be to vaccinate against strains that cause Chronic Diseases, Department of Microbiology Immunology and Infectious Dis- lethal visceral leishmaniasis (VL), for which the benefits of eases, Cumming School of Medicine, University of Calgary, Calgary, Alberta T2N 4Z6, Canada leishmanization may outweigh any risks. Cross-protection con- Received for publication March 31, 2015. Accepted for publication August 17, 2015. ferred by leishmanization against VL would suggest a common mechanism of resistance against Leishmania species that cause This work was supported by the Intramural Research Program of the National Insti- tute of Allergy and Infectious Diseases and by University of Calgary start-up funds different clinical diseases, and it would suggest that different (to N.C.P.). Leishmania species share a sufficient number of protective Ags to Address correspondence and reprint requests to Prof. Nathan C. Peters, Snyder In- warrant their use in pan-Leishmania vaccines (15, 16). However, stitute for Chronic Diseases, Department of Microbiology Immunology and Infec- evidence that L. major infection cross-protects against VL in tious Diseases, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive NW, Calgary, AB T2N 4Z6, Canada. E-mail address: [email protected] people is rare or difficult to interpret (17–23). The online version of this article contains supplemental material. Experimentally, two prior studies have investigated this question Abbreviations used in this article: BFA, brefeldin A; CL, cutaneous leishmaniasis; and found that leishmanization either provided no protection (24) dICS, direct intracellular staining; dLN, draining lymph node; GzB, granzyme B; i.d., or enhanced visceral infection (25) following L. infantum chal- intradermal(ly); LDA, limiting dilution analysis; LST, leishmanin skin test; VL, lenge. However, these studies employed BALB/c mice that are visceral leishmaniasis. susceptible to L. major infection due to a defect in the generation Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 of Th1 immunity, a condition not typically observed in people www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500752 The Journal of Immunology 3817 infected with L. major (26). In contrast, leishmanized C57BL/6 Parasite loads were determined by 2-fold serial dilutions in 96-well mice more closely replicate the immune status of leishmanized flat-bottom microtiter plates by overlaying 100 ml of the diluted tissue humans (11). Therefore, we employed an intradermal (i.d.) chal- suspension onto 50 ml NNN medium containing 20% defibrinated rabbit blood. The dilutions were made in duplicate. The plates were scored lenge model of visceral infection caused by L. infantum in C57BL/6 microscopically for growth and the number of parasites in each tissue mice leishmanized with L. major (27). We present evidence that was determined from the highest dilution at which parasites could be leishmanization provides robust protection and similar correlates grown out after 7–10 d incubation at 26˚C. In some experiments 25 mg/ml of protection against both cutaneous and visceral infection. hygromycin B (Sigma-Aldrich) was added to the media to discriminate between parasites used for live vaccination versus parasites used for Leishmanization may be a viable strategy for control of visceral challenge. disease. Restimulation of tissue-derived cells for cytokine analysis by Materials and Methods flow cytometry Parasites Tissue-derived cells were restimulated as described previously (11). Briefly, single-cell suspensions were incubated at 37˚C in 5% CO2 for L. major Friedlin strain was isolated from a patient who acquired his 12–14 h in flat-bottom 48-well plates with 0.5–1 3 106 T cell–depleted infection in the Jordan Valley (MHOM/IL/80/Friedlin). L. infantum (Miltenyi Biotec) naive spleen cells (APCs), with or without 50 mg/ml (MHOM/ES/92/LLM-320; isoenzyme typed MON-1) was isolated from freeze-thaw Leishmania Ag in a total volume of 1 ml. During the final 4– a patient with VL in Spain and was provided by Diane MacMahon-Pratt. 6hofculture,1mg/ml BFA (Sigma-Aldrich) was added. Cells were then L. infantum–RFP was generated as previously described (28). Parasites washed and labeled with LIVE/DEAD Fixable Aqua Cell Stain Kit were cultured in vitro at 26˚C in complete medium 199 supplemented (ThermoFischer Scientific) at a 1:500 dilution of the manufacturer with 20% heat-inactivated FCS (Gemini Bio-Products), 100 U/ml peni- suggested stock solution (Invitrogen) to exclude dead cells and anti–Fc cillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, III/II (CD16/32) receptor Ab (2.4G2), followed by PE-Cy7 anti-mouse Downloaded from 0.1 mM adenine (in 50 mM HEPES), 5 mg/ml hemin (in 50% trietha- CD4 (RM4-5), and in some experiments allophycocyanin–eFluor 780 nolamine), and 1 mg/ml 6-biotin. For L. infantum, the complete medium CD8a (53-6.7) for 20 min. In some experiments cells were stained with 199 was further supplemented with 2 mg/ml 6-biopterin (Sigma-Aldrich, PerCp-Cy5.5 anti-CD4 (RM4-5), FITC anti-CD44 (IM7), allophycocyanin– St Louis, MO). L. infantum and L. major infective-stage metacyclic eFluor 780 anti-Ly6C (HK1.4) and PE-Cy7 anti-CD62L (MEL-14) for promastigotes were isolated from stationary cultures (4–6 d old) by cen- 20 min. Cells were then fixed with BD Cytofix/Cytoperm (BD Bio- trifugation through a Ficoll-step gradient as described (29). For leishma- sciences) according to the manufacturer’s instructions and stained with nization, L. major metacyclic promastigotes were isolated by negative FITC anti–IFN-g (XMG1.2), PerCP–eFluor 710 anti–TNF-a (MP6- http://www.jimmunol.org/ selection of noninfective forms using peanut agglutinin (Vector Labora- XT22), and in some experiments allophycocyanin anti–TcR-b (145- tories) (30). 2C11) and/or PE anti-human granzyme B (GzB; GRB04, Invitrogen) Mice and/or V500 anti-CD3 (145-2C11). In some experiments, samples were treated with Foxp3 fixation/permeabilization buffer (eBioscience) per the Female C57BL/6 mice were obtained from Taconic. All mice were manufacturer’s instructions, and subsequently stained for 1 h at 4˚C with maintained in the National Institute of Allergy and Infectious Diseases PE anti–IFN-g (XMG1.2) and eFluor 660 anti-T-bet (eBio4B10) Abs. animal care facility under specific pathogen-free conditions. Isotype controls employed were rat IgG1 (R3-34) and rat IgG2b (A95-1 or eBR2a). All Abs were from eBioscience or BD Biosciences. Data Leishmanization and challenge were collected using FACSDiva software on a FACSCanto flow cytom- eter (BD Biosciences) and analyzed using FlowJo software (Tree Star). Leishmanized mice were generated by injecting 104 L. major metacyclic Forward scatter and side scatter widths were employed to exclude cell by guest on October 2, 2021 promastigotes s.c. in the hind footpad in a volume of 40 ml and used at 12– doublets from analysis. 20 wk after primary infection when footpad lesions had completely re- solved. Mice with a primary L. infantum infection where generated in the same manner. Naive mice, leishmanized mice, or L. infantum–infected Perfusion and in vivo staining of circulating cells 6 mice were challenged with 2 3 10 L. infantum metacyclic promastigotes In vivo labeling of circulating cells was done as described previously i.d. in the ear in a volume of 10 ml. In some experiments, mice were in- (32). Briefly, 0.6 mg anti–TCR-b BV421 (clone H57-597, BioLegend) 6 jected i.v. in the tail vein with 2 3 10 L. infantum metacyclic promastigotes wasinjectedi.v.in200ml PBS via the tail vein. Three minutes after the in a volume of 200 ml. injection, mice were sacrificed by isoflurane asphyxiation and perfused via intracardiac injection of 20 ml cold PBS. Liver perfusion was per- Processing of different sites of infection and parasite formed by injection of 6 ml cold PBS into the liver portal vein. Organs quantification were then harvested as described above. Following restimulation and b+ Mice were perfused via intracardiac injection of 20 ml cold PBS. Liver intracellular staining the frequency of TcR- cells labeled by i.v. ad- b perfusion was performed by injection of 6 ml cold PBS into the liver portal ministration of TcR- Ab was not reduced on T cells from the liver 6 b+ 6 b+ vein. The spleen and ear draining lymph node (dLN) were removed, cut with (30.0 13 TcR- before restimulation versus 29.6 12 TcR- after n tweezers, homogenized with a syringe plunger, and the cell suspension was restimulation and intracellular staining, = 14) and only slightly re- 6 b+ filtered through a 70-mm strainer. Livers were incubated for 45 min with 2 ducedoncellsfromthespleen(39.2 13 TcR- before restimulation 6 b+ ml DMEM containing 250 mg/ml Liberase TL purified enzyme blend versus 34.0 12 TcR- after restimulation and intracellular staining, n (Roche Diagnostics) and 10 mg/ml DNase. Liver cells were further purified =12). using a Percoll gradient (31). In experiments employing direct intracellular staining (dICS), organs were processed in media prewarmed to 37˚C Statistical analysis containing 20 mg/ml brefeldin A (BFA) and incubated at 37˚C and 5% CO2 Unless otherwise noted, data were compared using the Mann–Whitney U postprocessing for a total time in BFA of 4 h. Ear tissue was prepared as test. Comparisons between multiple groups were done using ANOVA with previously described (11). Briefly, ears were removed and placed in 70% a Holm–Sidek posttest for comparisons between multiple groups. All p ethanol for 2–5 min and then allowed to dry. Separated dorsal and ventral values are two-sided. Statistical calculations were done in GraphPad Prism sheets of ears were then incubated at 37˚C for 90 min in 1 ml DMEM 5.0c (http://www.graphpad.com). For figures depicting pooled data, error containing 160 mg/ml Liberase. Following Liberase treatment, tissue was bars represent the SEM. For figures in which data points represent indi- homogenized for 3.5 min in a Medicon using a Medimachine (Becton vidual replicates, error bars represent the SD of the mean. A p value ,0.05 Dickinson). The tissue homogenate was then flushed from the Medicon was considered significant. with 10 ml RPMI 1640 media containing 0.05% DNase and filtered using a 50-mm pore size cell strainer. In experiments employing dICS, BFA at Ethics statement 20 mg/ml was added to media prewarmed to 37˚C and the ear homogenate was returned to 37˚C and 5% CO2 postprocessing for a total time in BFA All animal experiments were performed under the LPD-68E Animal Study of 4 h. In some experiments mice were not perfused and RBCs were re- Protocol approved by the National Institute of Allergy and Infectious moved from the spleen and liver using ACK lysing buffer for 5 min at Diseases Animal Care and Use Committee using guidelines established by room temperature. Cells from each tissue were resuspended in complete the Animal Welfare Act and the Public Health Service Policy on Humane medium 199 with 6-biopterin. Care and Use of Laboratory Animals. 3818 CUTANEOUS LEISHMANIASIS PREVENTS VISCERAL LEISHMANIASIS

Results expansion between 1 and 14–21 d postinfection, peaking at 1737 in The course of L. infantum infection in C57BL/6 mice following the spleen and 5011 in the liver. Parasite numbers then underwent i.d. inoculation of the ear a gradual reduction between 21 and 63 d, and by day 84, parasites were no longer detected in the liver or spleen. This course of in- To study cross-protection against visceral infection, we challenged fection was in contrast to infection with L. major, which did not 3 6 naive or leishmanized C57BL/6 mice with 2 10 L. infantum expand in the viscera but established stable, chronic infections in parasites via the i.d. route in the ear. Although higher than the the skin and dLN following high-dose inoculation of the ear number of parasites deposited during natural sand fly transmission (Fig. 1D, 1E). (33), we employed 2 3 106 parasites because this was the lowest dose at which L. infantum parasites disseminated from the skin to Leishmanization with L. major provides protection against the spleen and liver in all naive mice tested (Fig. 1A). Although no visceral infection murine model replicates the development of progressive, fatal Twelve to 16 wk after leishmanization with L. major,micewere visceral leishmaniasis in people, the i.d. challenge model in challenged i.d. in one ear with L. infantum. Analysis of parasite C57BL/6 mice does allow for an immunological assessment of loads in the ear, dLN, liver, and spleen of leishmanized animals protective immunity in the skin, the physiological site of challenge revealed significant control of parasite numbers at all sites of (27). Additionally, the i.d. challenge model results in the delivery challenge and at all time points tested versus control animals, of low doses of parasites into the visceral organs, something that is with the exception of the dLN at 1 wk after challenge (Fig. 2A). also likely to occur during natural infection, and far different from At 3 wk after challenge, 40% (8 of 20) of the spleens and 55% the massive doses that are rapidly delivered to the viscera during (11 of 20) of the livers from leishmanized animals had unde- conventional i.v. challenge. tectable numbers of parasites and this increased to 88% (7 of 8) Downloaded from We initially determined using limiting dilution analysis (LDA) the at both sites by 6 wk after challenge. To compare the efficacy of course of L. infantum infection in naive C57BL/6 mice following prior infection with L. major versus L. infantum to protect against i.d. inoculation, because this has not been reported previously challenge with L. infantum, mice chronically infected with either (Fig. 1B, 1C). Following i.d. inoculation, parasites were found in strain were challenged with drug-resistant L. infantum parasites. the ear, dLN, liver, and spleen. In the ear and dLN, parasites initially At 1, 3, or 6 wk after challenge, LDA of parasites was performed increased in number followed by a decline between 7 and 42 d, at under drug pressure to eliminate the Leishmania employed for http://www.jimmunol.org/ which point parasite numbers stabilized for the remainder of the leishmanization. Leishmanization with L. major provided equiva- experiment. In the liver and spleen, parasites underwent significant lent or enhanced protection versus leishmanization with L. infantum by guest on October 2, 2021

FIGURE 1. The course of primary infection with L. infantum or L. major in C57BL/6 mice following i.d. inoculation of the ear. Naive mice were infected with the indicated doses of L. infantum (A) or with 2 3 106 L. infantum (B and C)orL. major (D and E) metacyclic pro- mastigotes in the left ear pinnae. Parasite loads in individual ears and dLNs (A, B, and D)or spleens and liver (A, C, and E) were determined at 1 wk (A) or the indicated time points (B–E) postinfection; n =4(A)orn =3(B–E) mice per time point. Representative results from two in- dependent experiments. Mean and SEM are shown. The Journal of Immunology 3819

A Ear Ear dLN 7 7 Infection *** **** **** 6 6 5 5 L. inf ** *** None 4 4 L. maj L. inf 3 3 2 2

1 Parasite laod (Log) 1 0 0 1w 3w 6w 1w 3w 6w Spleen Liver 4 ** 6 5 ** 3 ***

Parasite Load (Log) **** 4 ** * 2 3

2 1 1 Downloaded from 0 Parasite laod (Log) 0

1w 3w 6w 1w 3w 6w Weeks post challenge B Ear Ear dLN Spleen Liver Infection

*** 3 4 http://www.jimmunol.org/ 6 *** 6 $ ### #### #### # ## 3 L. inf 2 ## $ None 4 4 ### ### ## # 2 # L. maj L. inf 1 L. inf L. inf 2 2 1

0 0 Parasite Load (Log) 0 0 1w 3w 6w 1w 3w 6w 1w 3w 6w 1w 3w 6w Weeks post challenge

C by guest on October 2, 2021 Ear 106 105 Infection 104 103 L. maj L. inf p 102 L. maj L. maj 101 L. inf L. inf respective control Fold reduction vs. 100 1w 3w Weeks post challenge FIGURE 2. Primary infection with L. major protects against visceral infection with L. infantum. Naive mice or mice with a healed L. major (A–C)or L. infantum (B and C only) primary infection were challenged with 2 3 106 L. infantum (A–C)orL. major (C only) metacyclics in the ear. (A and B) Parasite loads in individual ears, dLNs, spleens, or livers at the indicated time points postchallenge. Data are the pool of five independent experiments including at least two time points per experiment. Mean and SD (A) or SEM (B) are shown. In (A), *0.01 , p , 0.05, **0.001 , p , 0.01, ***0.0001 , p , 0.001, ****p , 0.0001. In (B), ***0.0001 , p , 0.001, refer to differences between L. major/L. infantum and L. infantum/L. infantum; #0.01 , p , 0.05, ##0.001 , p , 0.01, ###0.0001 , p , 0.001, ####p , 0.0001 refer to differences between naive/L. infantum and L. major/L. infantum; $0.01 , p , 0.05 refers to differences between naive/L. infantum and L. infantum/L. infantum.(C) Mean (6SEM) fold parasite load reduction at the site of secondary challenge (ear) in mice with the indicated primary and secondary challenge infections versus the respective controls.

(Fig. 2B). This was most significant in the ear where the het- leishmanization with L. major provides protection against vis- erologous protection against L. infantum was similar to the level ceral infection. of protection against homologous challenge with L. major (Fig. + 2C). At all time points tested, with the exception of the 6 wk Leishmanization is associated with a rapid and robust CD4 time point in the liver when control mice are beginning to clear T cell response in the skin and visceral organs following parasites from this organ, protection was associated with sig- challenge nificantly greater numbers of leishmanized versus control mice IFN-g–producing CD4+ T cells mediate the protective response exhibiting sterilizing immunity in the spleen and liver as de- conferred by leishmanization against homologous challenge with termined by the inability to culture organisms from these tissues L. major (34, 35). Additionally, the rapidity of the CD4+ response (p # 0.036, Fisher exact test). As far as we know, these obser- is the clearest correlate of protection against sand fly–transmitted vations demonstrate the first experimental confirmation that disease (11, 12). Therefore, we analyzed IFN-g production by 3820 CUTANEOUS LEISHMANIASIS PREVENTS VISCERAL LEISHMANIASIS

CD4+ T cells at early time points following heterologous chal- mediating effector function in the organ. Whereas i.v. tail vein lenge with L. infantum and found increased frequencies of CD4+ challenge did not significantly change the data obtained from T cells with the capacity to make IFN-g in the skin, dLN, spleen, the spleen, there was a dramatic increase in the frequency of and liver of leishmanized versus control animals (Fig. 3). As IFN-g+CD4+ T cells from the liver that were i.v.2, suggesting previously reported (11), a rapid CD4+ T cells response in the skin that Ag load can drive the recruitment and/or retention of IFN-g– was observed within 3 d of i.d. challenge (Fig. 3B). We also ob- producing cells residing in the liver parenchyma (Fig. 4C, right served postchallenge responses in the viscera of leishmanized panel). Despite the fact that most IFN-g+CD4+ T cells associated mice that were significantly greater than prechallenge responses with the viscera were trapped i.v.+ circulating cells, dermal chal- startingatday3(liver)or1wk (spleen) postchallenge with lenge significantly increased the frequency of IFN-g+CD4+ cells L. infantum. This early response was similar to the response to that were i.v.2 versus prechallenge mice in both the spleen and homologous challenge with L. major (Supplemental Fig. 1), sug- liver (compare open circles in Fig. 4C). This was most dramatic in gesting that the early protective responses to L. infantum or the liver following tail vein challenge (Fig. 4C, right panel). To L. major are the same despite the different clinical outcomes correlate responses in the actual tissue with the reductions in of infection with these two parasites. Additionally, IFN-g produc- parasite numbers mediated by leishmanization observed in Fig. 1, tion was elicited equally well employing freeze-thawed Ag from we also determined the frequency of cytokine-producing cells L. infantum or L. major (ratio of response of L. major/L. infantum within the total, i.v.2, or i.v.+ CD4+ T cell populations following 1:0.95, n =4,p = 0.54, paired t test), suggesting these two para- challenge (Fig. 4D–F). In both the spleen and liver there was sites also share immunogenic MHC class II–restricted epitopes, as a significant increase in the frequency of IFN-g+ cells within suggested by work with the sterol 24-c-methyltransferase protein the i.v.2 population following i.d. challenge (Fig. 4E), dem- (36). onstrating that leishmanization mediates a rapid response in the Downloaded from

+ + visceral organs similar to that observed at the dermal site of chal- Localization of IFN-g CD4 T cells associated with the liver lenge. Analysis of i.v.+ cells revealed a trend toward increased and spleen employing in vivo i.v. staining frequencies of IFN-g+ cells following dermal challenge but this Leishmanized animals had low but detectable frequencies of did not reach significance. These observations demonstrate that CD4+ T cells with the capacity to make IFN-g upon stimulation leishmanization with L. major mediates an early CD4+ T cells in the spleen and liver prior to challenge (Fig. 4A). However, response in visceral sites of infection following i.d. challenge http://www.jimmunol.org/ similar cells were also found in the circulation, making it diffi- with L. infantum. cult to conclude whether these cells were residing in the tissue 2 prior to challenge or were simply trapped in the organ vascula- Intravenous , IFN-g–producing cells in the viscera following ture. Additionally, trapped circulating cells may contribute to the challenge are enriched for Ly6C expression increased frequencies of IFN-g+CD4+ T cells detected in the We have previously reported that leishmanization maintains high spleen and liver following challenge (Fig. 3B). Although we frequencies of short-lived CD44+CD62L2T-bet+Ly6C+CD4+ routinely perfuse our mice, and perfusion does reduce the fre- effector T cells that are required for protection against rein- quency of IFN-g+ cells in the liver and spleen (Supplemental fection with L. major in the ear dermis. These cells are not by guest on October 2, 2021 Fig. 2), recent observations have demonstrated that perfusion memory cells reactivated by secondary challenge, and although can be incomplete (32, 37). Therefore, to discern cytokine pro- they produce cytokine in vivo in an Ag-dependent manner, they duction from cells present in visceral tissues versus those trapped can be rapidly recruited to dermal sites of challenge in an Ag- in the vasculature, we in vivo labeled circulating cells prior to independent manner (35). Therefore, we wanted to determine euthanasia and perfusion (32). This technique labels cells on the whether IFN-g–producing cells in the viscera at 3 d following luminal side of the liver and spleen vasculature as well as cells in dermal challenge of leishmanized mice with L. infantum also the splenic red pulp. This analysis is important in the context of expressed the Ly6C+ effector T cell phenotype (Fig. 5). We vaccine studies to determine whether Ag-specific cells have ac- employed dICS to identify IFN-g–producing cells (35) (Fig. 5A). tually infiltrated infected tissues to mediate effector function Because dICS does not employ ex vivo Ag or pharmacological versus those that are circulating. Analysis of live CD4+ Tcells restimulation, assessment of Ly6C and T-bet expression using this from the blood following in vivo labeling revealed .99% were methodology likely reflects patterns of in vivo expression. Non- i.v.+ as expected (Fig. 4B). In the liver and spleen we found both circulating i.v.2CD44+CD62L2CD4+ T cells that expressed Ly6C i.v.2 and i.v.+ cells, despite whole-body and liver-specific per- were T-bethigh, as described previously (35) (Fig. 5B), and were fusion (Fig. 4B). We then determined the frequency of cytokine- highly enriched for IFN-g–producing cells compared with Ly6C2 producing cells that were in the tissue versus those trapped in the cells (Fig. 5C). Additionally, the frequency of T-bet+Ly6C+– vasculature of the ear, spleen, and liver prior to and at 1 wk expressing cells was significantly higher among IFN-g–producing following i.d. challenge, the peak of the CD4+ IFN-g Tcellre- i.v.2CD44+CD62L2CD4+ T cells versus IFN-g nonproducing sponse. We also employed i.v. challenge with 2 3 106 L. infantum, i.v.2CD44+CD62L2CD4+ T cells (Fig. 5D). In contrast, IFN-g which delivers large numbers of parasites directly into the visceral production in naive mice challenged with L. infantum was very organs, particularly the liver (27), to act as a positive control for low, and no significant difference was observed between Ly6C+ Ag-driven recruitment and/or retention of IFN-g+CD4+ cells in the and Ly6C2 cells. These data demonstrate that, similar to L. major spleen and liver. Both prior to and following dermal challenge, the challenge of the ear, IFN-g production is highly associated with vast majority of IFN-g+ cells in the ear were i.v.2 (Fig. 4C, left Ly6C-expressing CD4 T cells in the viscera at acute time points panel), demonstrating that these cells were residing in the tissue. following challenge with L. infantum. In contrast, most IFN-g+CD4+ cells in the spleen and liver were + 2 + , Following challenge, responding CD8 T cells are i.v. , despite perfusion (i.v. versus i.v. , p 0.001), demon- + 2 strating that these cells were trapped, circulating cells. This predominantly GzB but IFN-g suggests that most Leishmania-specific cytokine production Whereas Th1 CD4+ T cells are associated with mediating pro- attributed to cells associated with the visceral organs in Fig. 3B tection against primary and secondary infection with L. major is due to cells trapped in the vasculature, not cells that are actually (35, 38), the role of CD8+ T cells in leishmaniasis remains The Journal of Immunology 3821 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Kinetic of IFN-g–producing CD4+ T cells in the skin and viscera after challenge infection with L. infantum. At the indicated time post- challenge, cells from mice as shown in Fig. 2A were restimulated in vitro with APCs plus a mixture of L. major and L. infantum Ag and analyzed by flow cytometry. (A) Representative intracellular staining contour plots for cytokine-producing CD4+TCRb+ T at 1 wk postchallenge. (B) Kinetic of IFN-g production by CD4+TCRb+ T cells in ears, dLNs, spleens, and livers. *0.01 , p , 0.05, **0.001 , p , 0.01, ***0.0001 , p , 0.001 refer to differences between leishmanized unchallenged mice and L. major/L. infantum; #0.01 , p , 0.05, ##0.001 , p , 0.01, ###0.0001 , p , 0.001, ####p , 0.0001 refer to differences between naive/L. infantum and naive control mice; $$0.001 , p , 0.01, $$$0.0001 , p , 0.001, $$$$p , 0.0001 refer to differences between naive/L. infantum and L. major/L. infantum; n = 4–5 mice per group per time point. Data are the pool of five independent experiments including at least two time points (day 3, 1 wk, and/or 3 wk) per experiment. 3822 CUTANEOUS LEISHMANIASIS PREVENTS VISCERAL LEISHMANIASIS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. In vivo i.v. staining reveals localization of IFN-g+CD4+ T cells in the spleen and the liver. (A) Prechallenge production of IFN-g by CD4+ TCRb+ T cells from different organs of uninfected or leishmanized mice following ex vivo Ag restimulation as described in Fig. 3. (B) Representative histograms of i.v staining with anti–TCRb-BV421 on CD4+ T cells in the blood, spleen and liver. (C) Proportion of i.v.2 or i.v.+ cells within the IFN-g+ CD4+ T cell population (following ex vivo Ag restimulation) from the ear, spleen, or liver of leishmanized mice following no challenge (Lm-⃠), i.d. challenge in the ear (Lm-Li (Ear)), or i.v. challenge in the tail vein (Lm-Li (Tail Vein) with 2 3 106 L. infantum metacyclics. **0.001 , p , 0.01, ***0.0001 , p , 0.001, ****p , 0.0001 refer to differences between Lm-⃠ and Lm-Li (Ear); #0.01 , p , 0.05, ####p , 0.0001 refer to differences between Lm-⃠ and Lm-Li (Tail Vein); $$$$p , 0.0001 refers to differences between Lm-Li (Ear) and Lm-Li (Tail Vein). (D–F) Percentage of IFN-g+ cells in the total CD4+ T cell population (D), the i.v.2CD4+ T cell population (E), or the i.v.+CD4+ T cell population (F). One week after challenge data point is shown. Data are the pool of three independent experiments employing four to five mice per experiment (two experiments employed tail vein challenge). Li, L. infantum; Lm, L. major. The Journal of Immunology 3823 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. i.v.2CD44+CD62L2Ly6C+CD4+ T cells in the viscera of leishmanized mice are enriched for IFN-g–producing cells following challenge. Naive or leishmanized animals were challenged with L. infantum as described in Fig. 2. On day 3 postchallenge CD4+TcRb+ T cells were analyzed for CD44, CD62L, Ly6C, T-bet, and IFN-g expression employing dICS. (A) Representative dot plots of IFN-g production by i.v.2CD4+ T cells. (B) Rep- resentative histogram of T-bet expression by the indicated cell populations in the spleen. (C) Percentage of IFN-g+ cells within live i.v.2CD4+CD44+ CD62L2Ly6C+ or live i.v.2CD4+CD44+CD62L2Ly6C2 populations. (D) Percentage of Ly6C+T-bet+ cells within the live i.v.2CD4+CD44+CD62L2IFN-g+ or live i.v.2CD4+CD44+CD62L2IFN-g2 population. In (C) and (D), n = 4 mice/group. Data are representative of two independent experiments. *0.01 , p , 0.05, **0.001 , p , 0.01, ***0.0001 , p , 0.001, ****p , 0.0001. controversial and is dependent on the dose, strain, and infection Employing in vivo labeling of circulating cells, we detected an setting (primary or secondary) (1, 38–41). In visceral infection increase in the frequency of GzB+CD8+ T cells in the viscera with L. donovani,CD8+ T cells have been shown to be both following challenge, although this did not reach significance protective and dispensable (40, 42). We determined IFN-g and (Supplemental Fig. 4). GzBproductionbyCD8+ T cells following challenge with L. infantum and, similar to observations in BALB/c mice (24), Discussion found a very low frequency of IFN-g+ cells regardless of the Our results demonstrate for the first time, as far as we know, that site of analysis (Fig. 6). In contrast, high frequencies of cells leishmanization protects against heterologous visceral infection. were found to be GzB+. Similar observations were made fol- Protection was associated with rapid activation and recruitment of lowing challenge with L. major (Supplemental Fig. 3). The IFN-g–producing Th1 cells at both the cutaneous site of challenge postulated role of GzB in leishmaniasis is evolving. Granzyme and visceral sites of infection, demonstrating that protection 2 2 A3B / double knockout C57BL/6 mice are completely re- conferred by leishmanization is not site specific. Similar to what sistant to L. major infection (43) whereas CD8+GzB+ T cells we have previously reported for L. major challenge in the skin, have been associated with pathology, not parasite killing, IFN-g production was highly associated with CD44+CD62L2 during cutaneous L. braziliensis infection (41, 44, 45). Despite T-bet+Ly6C+CD4+ T cells within the i.v.2 population in the vis- the high frequency of GzB+CD8+ cells we were unable to detect cera. At early time points following challenge these cells are GzB by ELISA following Ag stimulation (data not shown). recruited, infection-dependent, pre-existing effector T cells, not 3824 CUTANEOUS LEISHMANIASIS PREVENTS VISCERAL LEISHMANIASIS Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. Kinetics of GzB-producing CD8+ T cells in the skin and viscera after challenge with L. infantum. At indicated times postchallenge, cells were restimulated in vitro with APCs plus a mix of L. major and L. infantum Ags and analyzed by flow cytometry. (A) Representative intracellular staining contour plots for GzB- and IFN-g–producing CD8+TCRb+ T cells at 3 d postchallenge. (B) Kinetic of GzB production by CD8+TCRb+ T cells in the ears, dLNs, spleens, and livers. *0.01 , p , 0.05, **0.001 , p , 0.01 refer to differences between leishmanized unchallenged mice and L. major/ L. infantum; #0.01 , p , 0.05, ##0.001 , p , 0.01 refer to differences between naive/L. infantum and naive control mice; $$$$p , 0.0001 refer to differences between naive/L. infantum and L. major/L. infantum; n = 4–5 mice per group per time point. Data are the pool of three (ears) or four (other sites) independent experiments. The Journal of Immunology 3825 memory cells reactivated by challenge (35). Our observations following needle challenge has proven to be an informative cor- suggest that the correlates of protective immunity against cuta- relate of protection against cutaneous disease following challenge neous leishmaniasis (CL) and VL may be sufficiently similar to with L. major–infected sand flies (11, 12). warrant a pan-Leishmania vaccine provided the vaccine can rep- Our observations suggest that most cytokine production typically licate the correlates of immunity observed in leishmanized indi- associated with adaptive T cell immunity in the spleen and liver is viduals (11, 12, 35). Our data also provide the best evidence to from circulating cells trapped in the vascular or the splenic red pulp date that heterologous immunity can be identical, or even superior, at the time of euthanasia. Despite this observation, we were able to to homologous immunity. employ a combination of in vivo labeling of circulating cells and The controlled trials demonstrating the efficacy of leishmanization prechallenge leishmanized mice to demonstrate that leishmani- in humans is restricted to protection against CL caused by L. major or zation does mediate a rapid Leishmania-specific immune response . However, epidemiological studies suggest prior in visceral tissue in response to dermal challenge with L. infantum. infection of humans with L. major may provide cross-protection The use of the in vivo labeling technique would appear to be against VL caused by L. donovani. Two longitudinal studies of VL critical in future studies to determine the relative contribution of in Sudan demonstrated that despite the presence of both leishmanin circulating cells versus tissue-resident cells to total cytokine skin test (LST)–positive and –negative individuals at the beginning of production. Importantly, note that even before challenge, we found each study, all cases of VL were reported among the LST-negative low frequencies of i.v.2 cells with the capacity to make IFN-g group (17, 18). Because LST positivity was highly associated with in the uninfected ear, non-dLN, blood, liver and spleen of prior residence in an L. major endemic area, the authors hypothesized leishmanized mice, similar to what we have reported previously that prior infection with L. major provides protection against VL (35). Whether the i.v.2IFN-g+CD4+ T cells in the viscera are caused by L. donovani. Related observations in Sri Lanka reported tissue-resident memory T cells, as has been reported for the lung Downloaded from that districts with high levels of CL caused by a naturally attenuated (58), skin (59), and vaginal mucosa (60), or patrolling cells, as has strain of L. donovani are almost devoid of VL (19). Experimentally, also been reported for the skin (61), is unknown. Whereas pre- susceptible BALB/c mice infected with the attenuated L. donovani existing Ag-specific T cells are not required for the recruitment of strain had significantly reduced parasite burden in the liver upon highly protective Leishmania-specific T cells into a site of non- challenge with the L. donovani strain responsible for Sri Lankan VL specific tissue damage (35), this does not preclude the possibility (46). In a vervet monkey model of disease, Gicheru et al. (47) re- that they may enhance protection, regardless of their patrolling or http://www.jimmunol.org/ ported the inverse result, that subclinical infection with L. donovani resident nature as recently shown (59). Extensive studies have provided complete protection against cutaneous leishmaniasis fol- identified a role for pre-existing CD8+ tissue-resident memory lowing experimental challenge with L. major. T cells in protective immunity (62). We found strong evidence for Experimental cross-protection mediated by infection (24, 25, 48– the existence of i.v.2, resident, Leishmania-specific GzB+CD8+ 54) as well as T cell cross-reactivity with Ags from different T cells in the viscera of leishmanized mice prior to challenge. The Leishmania species (15, 16) have been studied extensively in animal role of these cells in protection will be the topic of future research. models, primarily in an effort to define proteins that could be used in Our data reinforces the rationale for reintroducing the practice of apan-Leishmania vaccine. Genome sequencing has also revealed leishmanization under conditions where the benefits, such as pro- by guest on October 2, 2021 that 90% of the genome is conserved between L. major and tection against fatal visceral disease, are deemed to outweigh the L. infantum, and 99% of the genes are syntenic (55, 56), suggesting risks associated with administering live L. major. Numerous ge- a strong likelihood of shared Ags. Remarkably, only two experi- netically modified or avirulent Leishmania strains have been studied mental studies have investigated the cross-protection mediated by in an effort to replicate the protective effect of leishmanization primary infection with L. major against subsequent visceral infection while reducing or controlling for the virulence of the parasite (52, (24, 25). These studies employed BALB/c mice chronically infected 63–68). How effective these modified strains are compared with with L. major and showed no protection against either i.v. or i.d. leishmanization with wild-type L. major has not been addressed, challenge with L. infantum. In contrast, we were able to demonstrate with a single exception (68). Past observations (34, 35, 69, 70) the protective effect of leishmanization against visceral infection suggest that attenuation resulting in sterile clearance rather than employing C57BL/6 mice with a healed L. major infection, which persistence will compromise long-term protection, especially more closely replicates the immune status of leishmanized humans. against the more stringent conditions of infected sand fly challenge. Our observations are limited to an assessment of protection against The ideal situation would be to leishmanize with a parasite that infection, not disease, because mice infected i.d. with visceralizing minimizes pathology but also persists over the long term. An ad- strains of Leishmania produce minimal pathology in the viscera. ditional drawback of leishmanizing against VL is the wisdom of Additionally, the i.d. model of infection does not establish long-term introducing L. major in areas where the parasite is not endemic, infection in naive mice as determined by the detection of parasites in such as northeast India. However, L. major strains that establish the liver and spleen by LDA at 12 wk postinfection (Fig. 1). Al- persistent infection in the mammalian host but cannot produce though this may be viewed as a limitation of the model, the use of transmissible infections in the sand fly midgut, such as various lpg the more conventional high-dose i.v. challenge model would bypass knockout parasites, have already been developed that would address the potentially important initial step of parasite deposition in the these concerns (67, 71–73). skin, the natural site of infection. Additionally, note that in humans the immunobiology of asymptomatic infection with visceralizing Acknowledgments parasites is understudied, and the scarcity of parasites in the viscera We thank Kim Beacht, Melanie Faivre-Charmoy, and Flavia L. Ribeiro- mayinfactbeacommonfindinginthefield(57). Gomes for assistance with experiments. We especially thank Kristin Although the ideal challenge model would employ exposure to Anderson and David Masopust (University of Minnesota) and Dan Barber the bites of L. infantum–infected sand flies, we have yet to es- (National Institute of Allergy and Infectious Diseases) for help with the i.v. tablish a reliable sand fly transmission model of L. infantum in our staining protocol. laboratory. Rather, we employed relatively early time points, that is, 1 wk after needle challenge, to determine protective immunity Disclosures in the viscera, as early control of parasite numbers in the skin The authors have no financial conflicts of interest. 3826 CUTANEOUS LEISHMANIASIS PREVENTS VISCERAL LEISHMANIASIS

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