CD8؉ CTLs Are Essential for Protective Immunity Against Encephalitozoon cuniculi Infection1

Imtiaz A. Khan,2* Joseph D. Schwartzman,† Lloyd H. Kasper,* and Magali Moretto*

Encephalitozoon cuniculi is a protozoan parasite that has been implicated recently as a cause of in im- munocompromised individuals. Protective immunity in the normal host is T cell-dependent. In the present study, the role of individual T cell subtypes in immunity against this parasite has been studied using knockout mice. Whereas CD4؊/؊ animals resolved the infection, mice lacking CD8؉ T cells or perforin gene succumbed to parasite challenge. The data obtained in these studies suggest that E. cuniculi infection induces a strong and early CD8؉ T response that is important for host protection. The CD8؉ T cell-mediated protection depends upon the CTL activity of this cell subset, as the host is rendered susceptible to infection in the absence of this function. This is the first report in which a strong dependence upon the cytolytic activity of host CD8؉ T cells has been shown to be important in a parasite infection. The Journal of Immunology, 1999, 162: 6086–6091.

icrosporidia are a group of eukaryotic, obligate intra- endothelial cells, fibroblasts, and macrophages in wide variety of cellular parasites that infect an extremely wide range , including rabbits, rodents carnivores, monkeys, and hu- M of hosts within the animal kingdom (1). They are mans (15). In an experimental model, normal mice infected with E. unique enough to be placed in a separate phylum, Microspora, (2) cuniculi usually express few clinical signs of disease (16). Con- and are characterized by a polar filament that is used to inject versely, immunodeficient hosts such as athymic or SCID mice de- sporoplasm in the host cell (3). Species of that infect velop lethal disease after experimental infection (17, 18). These mammals are unicellular, Gram-positive organisms 0.5 ϫ 1–4 ␮m animals reveal numerous microsporidia in visceral and parietal in diameter (4). Classification is based on size, nuclear arrange- peritoneum as well as in the liver and spleen. Previous studies have ment, and mode of division and association of proliferative forms shown that T cells are responsible for the prevention of lethal within the host cell. disease. An adoptive transfer of enriched spleen cells from an in- Of the 80 genera in the phylum Microspora, several have been fected host with normal immunity protects athymic or SCID mice demonstrated in human disease (5). Symptoms due to infection are against E. cuniculi challenge (19, 20). However, none of these found in HIV-infected or other immunocompromised individuals studies have delineated the role of individual T cell subtypes in (6). Enterocytozoon bieneusi, which is the most common micro- immunocompetent E. cuniculi-infected animals. In the present sporidian observed in AIDS patients, infects enterocytes of the study, we observed significant increases in the CD8ϩ T cell pop- bowel and causes diarrhea (7). Encephalitozoon hellem and En- ulation during E. cuniculi infection. Mice lacking CD8ϩ T cells cephalitozoon intestinalis, which are closely related to Encepha- were highly susceptible to infection and developed evidence of litozoon cuniculi, are both reportedly associated with disseminated infection in several tissues. infection during HIV infection (2). E. cuniculi, which was ob- served previously in laboratory animals, is considered to be a zoo- Materials and Methods notic infection (6). Several cases of HIV-infected individuals suf- Mice fering from the complications due to E. cuniculi infection have Ϫ/Ϫ 3 been documented recently (8, 9). HIV-infected patients with E. A breeding pair of CD8 knockout (KO) animals on a C57BL/6 back- ground was kindly provided by Dr. T. W. Mak (Amgen Institute, Ontario, cuniculi infection have presented with renal failure, pneumonitis, Canada). Animals were bred under approved conditions at the Animal Re- sinusitis, keratopathy (10, 11), granulomatous liver necrosis (12), search Facility at Dartmouth Medical School. CD4Ϫ/Ϫ and perforinϪ/Ϫ and peritonitis (13). In a recent report, autopsy findings in a patient (PFϪ/Ϫ) mice having similar genetic lineage were obtained from The Jack- with AIDS showed disseminated E. cuniculi infection involving son Laboratory (Bar Harbor, ME). Age- and sex-matched C57BL/6 mice the brain (8). were used as wild-type (wt) controls. Little is known regarding host immunity to microsporidia, in- Parasite and infection cluding E. cuniculi. E. cuniculi was the first mammalian micro- sporidian successfully grown in vitro (14). It infects epithelial and A rabbit isolate of E. cuniculi organisms was kindly provided by Dr. Eliz- abeth Didier of the Tulane Regional Primate Center. The parasites were maintained by continuous passage in a rabbit cell line (RK-13 cells) obtained from the American Type Culture Collection (Manassas, VA). The Departments of *Medicine and Microbiology and †Pathology, Dartmouth Medical ϫ 7 School, Lebanon, NH 03756 experimental animals were infected by i.p. injections of 1 10 spores. Received for publication December 1, 1998. Accepted for publication February 17, 1999. Histopathology The costs of publication of this article were defrayed in part by the payment of page Tissues from infected PF and CD8 KO animals as well as parental control charges. This article must therefore be hereby marked advertisement in accordance animals were fixed in 10% buffered formalin and processed for 5-␮m his- with 18 U.S.C. Section 1734 solely to indicate this fact. tological sections, which were stained with hematoxylin and eosin. 1 This work was supported by National Institutes of Health Grant AI43693. 2 Address correspondence and reprint requests to Dr. Imtiaz A. Khan, Department of Medicine, Dartmouth Medical School, HB 7506, One Medical Center Drive, Leba- 3 Abbreviations used in this paper: KO, knockout; PF, perforin; wt, wild type; non, NH 03756. E-mail address: [email protected] CD62L, CD62 ligand; p.i., postinfection.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 6087

Table I. Phenotypic expression of splenocytes following E. cuniculi infectiona

CD4ϩ CD8ϩ NKϩ Days p.i. Treatment % Positive Abs. No. % Positive Abs. No. % Positive Abs. No.

10 Uninfected 18.95 Ϯ 0.07 0.52 Ϯ 0.01 11.95 Ϯ 0.63 0.31 Ϯ 0.02 5.65 Ϯ 0.50 0.20 Ϯ 0.04 Infected 14.25 Ϯ 2.19 0.63 Ϯ 0.02 18.65 Ϯ 2.56 0.76 Ϯ 0.12 12.50 Ϯ 0.06 0.53 Ϯ 0.08 17 Uninfected 20.00 Ϯ 2.40 0.62 Ϯ 0.02 12.00 Ϯ 0.14 0.29 Ϯ 0.04 8.40 Ϯ 0.56 0.22 Ϯ 0.03 Infected 22.15 Ϯ 1.13 0.78 Ϯ 0.04 31.84 Ϯ 1.85 1.31 Ϯ 0.07 11.65 Ϯ 0.50 0.45 Ϯ 0.01 24 Uninfected 18.20 Ϯ 0.14 0.62 Ϯ 0.03 11.55 Ϯ 2.19 0.34 Ϯ 0.03 6.55 Ϯ 0.35 0.18 Ϯ 0.05 Infected 19.25 Ϯ 2.33 0.76 Ϯ 0.09 17.45 Ϯ 0.78 0.69 Ϯ 0.03 5.20 Ϯ 0.14 0.21 Ϯ 0.04

a C57BL/6 mice (n ϭ 3/group) were infected i.p. with 1 ϫ 107 spores of E. cuniculi. Splenocytes were isolated and pooled at various timepoints p.i. The cells were phenotyped for the expression of CD4, CD8, and NK receptors by direct immunofluorescence using FACS. Data are represented as mean Ϯ SD of two similar experiments. Abs. No. ϭ Absolute number of phenotypic cells ϫ 108.

Phenotypic analysis crease in the cell type over the uninfected controls was observed. ϩ Following euthanasia, the spleens from infected animals were removed and The CD8 T cell population remained elevated at day 24 p.i, sug- homogenized in a petri dish. The contaminating RBCs were lysed in RBC gesting that these cells may be involved in a long-term immune lysis buffer (Sigma, St. Louis, MO). Cells were washed and suspended in response. The population of NK cells also rose during early infec- 3% PBS/BSA. Splenocytes were analyzed for cell phenotype by FACS tion. At day 10 p.i., a Ͼ2-fold increase in both the percentage and (Becton Dickinson, Mountain View, CA) using a direct immunofluores- absolute number of NK cells was observed. However, by day 17 cence assay. Cells (1 ϫ 106/ml) were incubated with 1 ␮g of FITC-labeled anti-CD4ϩ, anti-CD8ϩ, or anti-NK1.1 at a 1/100 dilution (PharMingen, postchallenge, the absolute number of the NK cell population was San Diego, CA) in 3% BSA/PBS. After1hofincubation at 4°C, the cells close to uninfected controls. There was no significant change in the were washed several times in buffer, fixed in 1% methanol free formalde- CD4ϩ T cell population during the course of infection. hyde, and stored cold for FACS analysis. CD8ϩ T cell subset is activated early during E. cuniculi Purification of CD8ϩ T cells infection CD8ϩ T cells from the whole splenocyte population was separated by The data obtained from the above studies demonstrated an early microbeads (Miltenyi Biotec, Auburn, CA) as described previously (21). ϩ The separation procedure was conducted as recommended by the manu- induction of the CD8 T cell response during E. cuniculi infection. facturer. The purity of the separated cells was Ͼ95% as determined by We subsequently determined the earliest timepoint after infection ϩ FACS analysis. Purified CD8 T cells were assayed for CD69, CD62 li- at which CD8ϩ T cell activation begins. For this purpose, purified gand (CD62L), and CD44 expression by direct immunofluorescence. The CD8ϩ T cells from infected animals were stained for CD69, which cells were incubated with FITC-conjugated Ab to CD69, CD62L, or CD44 (PharMingen) and analyzed by FACS as described above. is a surface marker that is rapidly induced in activated T cells (24). As shown in Table II, a significantly increased percentage of Cytotoxic assay CD8ϩ T cells expressing CD69 was observed as early as day 4 p.i. A CTL assay was performed to detect the cytotoxic activity of the spleno- (21 Ϯ 7) compared with uninfected controls (6 Ϯ 3) ( p ϭ 0.02). ϩ cytes from infected animals. The assay was carried out using a protocol CD8 T cells from the infected animals continued to show higher standardized previously in our laboratory (22). Briefly, peritoneal macro- CD69 expression at later timepoints. By day 18 p.i., a greater per- phages from thioglycolate-treated mice were collected, washed, and dis- centage of CD8ϩ T cells exhibited increased expression of CD44 pensed at a concentration of 5 ϫ 104 cells/well in U-bottom, 96-well plates. high ϭ After overnight incubation, the cells were infected with 2 ϫ 105 spores of (CD44 ), a surface maker present on the activated cells ( p low E. cuniculi per well for 48 h. The wells were washed extensively with PBS 0.01). Conversely, low expression of CD62L (CD62L ), a sur- to clear extracellular parasites. Macrophages were labeled with 51Cr (0.5 face marker that is frequently identified with naive or uncommitted ␮Ci/well) for2hat37°C. Macrophages were washed five times with PBS T cells, was observed at day 18 postchallenge. and incubated with cultured spleen cells at various E:T ratios in a final ␮ volume of 200 l of culture medium. The microtiter plates were centri- CD8ϩ T cells play an important role in protective immunity fuged at 200 ϫ g for 3 min and incubated at 37°C for 4 h. Samples (100 ␮l) were removed and assayed for released cpm by scintillation counting. against E. cuniculi The percentage of lysis was calculated as follows: ([mean cpm of test Phenotypic analysis of splenocytes from infected animals sug- sample Ϫ mean cpm of spontaneous release]/[mean cpm of maximal re- ϩ lease Ϫ mean cpm of spontaneous release])/100. gested an important function for CD8 T cells during E. cuniculi infection. The role of individual T cell subtypes in immunity Statistical analysis against the parasite was studied using gene KO animals. The mice Statistical analysis of the data was performed using a two-sampled Student t test (23). Table II. Phenotypic changes on CD8ϩ T cells following E. cuniculi Results infectiona E. cuniculi infection induces a preferential expansion of CD8ϩ T cells Percentage of Positive Cells Days To determine which immune cells were responding to E. cuniculi Postinfection CD69 CD44high CD62Llow infection, a phenotypic analysis of the splenocytes from infected animals was performed. Spleen cells were isolated at days 10, 17, Day 0 6 Ϯ 355Ϯ 679Ϯ 8 Ϯ Ϯ Ϯ and 24 postinfection (p.i) and analyzed for the expression of var- Day 4 21 7493806 Day 9 22 Ϯ 661Ϯ 10 70 Ϯ 14 ious immune cell phenotypes. As shown in Table I, the increase in Day 18 20 Ϯ 12 67 Ϯ 662Ϯ 1 ϩ the absolute numbers of the CD8 T cell population became ap- ϩ ϩ a Purified CD8 T cells (5 ϫ 105) were stained for different phenotypic markers parent at day 10 p.i. The rise in the CD8 T cell population was directly by FITC-labeled Abs (PharMingen) and analyzed by FACS. Cells were further enhanced by day 17 p.i. At this timepoint, a Ͼ3-fold in- Ͼ95% pure, and data are shown as mean Ϯ SD of three separate experiments. 6088 CTL RESPONSE DURING E. cuniculi INFECTION

FIGURE 1. Survival of gene KO mice from E. cuniculi challenge. Female CD4 and CD8 KO mice on a C57BL/6 background that were 5–6 wk of age as well as parental wt mice (n ϭ 6/gp) were infected i.p with 1 ϫ 107 spores of E. cuniculi. Mortality was monitored on daily basis. The study was performed three times with similar findings.

were infected with E. cuniculi spores, and survival was monitored. dence of mild inflammation, most prominently in the liver. Small All of the CD8Ϫ/Ϫ mice challenged with the parasites died be- lymphocytic collections were noted in the liver, with rare poly- tween days 15 and 20 p.i. (Fig. 1). Just before death, these mice morphonuclear leukocytes (Fig. 3A). Evidence of hepatocyte ne- ϩ became lethargic and developed ascitis. Mice lacking CD4 T crosis was rare. The morphology of the spleen was preserved, and cells survived the infection until termination of the experiment, there was little indication of inflammation or the destruction of similar to parental wt controls. None of these animals developed other organs (Fig. 3D). Very rare mononuclear cells infected with clinical signs of disease (lethargy, ruffled skin, or the development microsporidia were evident by hematoxylin and eosin staining, and of ascitis). microsporidial spores were visualized by birefringence when CD8ϩ T cell immunity is dependent upon PF-mediated cytolysis viewed by polarization microscopy (data not shown). Ϫ/Ϫ ϩ In contrast, infected CD8 animals showed prominent hepatic CD8 T cells play an important role in intracellular infections by and splenic parasite loads. Multiple nodules of acute and chronic their ability to produce protective cytokines or lyse infected targets inflammation exhibiting hepatocyte necrosis and enlarged mono- (25–28). The major killing mechanism exhibited by CD8ϩ T cells nuclear cells with abundant microsporidial spores were scattered in many intracellular infections is via the PF pathway (29). To throughout the liver parenchyma (Fig. 3C). The spleen showed determine whether PF is important in the natural immunity against areas of acute inflammation and necrosis in the white pulp, with E. cuniculi, mice lacking this gene were infected i.p. with 1 ϫ 107 spores. Mice deficient in the PF gene succumbed to infection al- many enlarged mononuclear cells filled with microsporidia (Fig. most at the same time as CD8Ϫ/Ϫ animals (Fig. 2). Similar to 3F). Other organs showed widely scattered areas of less intense CD8Ϫ/Ϫ mice, these animals developed ascitis and became inac- inflammation (data not shown). Microsporidia were not detected in tive. As observed earlier, none of the parental mice died or devel- sites other than the liver and spleen. Ϫ/Ϫ oped illness throughout the course of the experiment. Infected mice with the PF genotype showed a pattern similar to the CD8Ϫ/Ϫ mice. The parasite load and inflammatory response Histopathological analysis were high in both the liver and spleen (Fig. 3, B and E). Other Tissues from infected KO and parental wt animals were isolated organs were much less affected, and parasite-infected cells were and subjected to histopathological analysis at day 14 postchal- only evident in areas of necrosis and inflammation within the liver lenge. Infected C57BL/6 parental animals showed scattered evi- and spleen.

FIGURE 2. Survival of PFϪ/Ϫ mice against E. cuniculi infection. PFϪ/Ϫ and wt C57BL/6 mice of 5–8 wk of age (n ϭ 6/group) were infected with E. cuniculi as described in Ma- terials and Methods. The animals were evalu- ated daily for signs of morbidity (lethargy, ruf- fled skin, and ascitis) and mortality. The experiment was performed three times with similar results. The Journal of Immunology 6089

FIGURE 3. Photomicrographs of livers (A–C) and spleens (D–F)ofwt (A and D), PFϪ/Ϫ (B and E), and CD8Ϫ/Ϫ (C and F) mice infected with E. cuniculi. Arrows indicate cystic structures filled with microsporidial forms. The bars in A, B, and C equal 25 ␮; the bars in D, E, and F equal 100 ␮.

E. cuniculi-infected mice develop a cytotoxic T cell response lier (Fig. 4, B and C). Immune spleen cells were unable to lyse the The data obtained from the PF KO animals suggested that a cy- infected macrophages from nonsyngeneic BALB/c mice (data not totoxic T cell response in E. cuniculi-infected animals could be an shown). These findings indicate that the development of an MHC- important component of the protective immune system. To deter- restricted CTL response may play a role in the protection of the mine whether this is the case, an in vitro CTL assay was performed host against the parasite. using splenocytes from E. cuniculi-infected animals. Spleen cells Cytotoxic response during E. cuniculi infection is mediated by were harvested and cultured in the presence of irradiated spores for ϩ 5 days. The viable cells were isolated by Ficoll and incubated with CD8 CTLs infected macrophages at various E:T ratios. Ag-stimulated spleen Although cytolysis is a function of CD8ϩ T cells, other cell types, cells from animals infected on days 0 and 7 failed to exhibit a such as NK cells, can also exhibit this property (30). To verify that cytolytic effect (Fig. 4A). However, 50–60% target cell lysis was this was not the case, mice were infected i.p. with E. cuniculi as observed with spleen cells from animals infected 17–24 days ear- described earlier. At day 17 p.i., the splenocytes from the infected

FIGURE 4. E. cuniculi infection in mice generates a CTL response. Pooled splenocytes (n ϭ 3/group) from 5- to 8-wk-old female C57BL/6 mice infected with E. cuniculi were isolated after 10 (A), 17 (B), and 24 (C) days p.i. Cells were cultured in vitro with 5 ϫ 103 irradiated spores. After 5 days of incubation, viable cells were separated and cultured with 51Cr-labeled macrophages infected with E. cuniculi or uninfected targets at various E:T ratios. At 4 h after incubation, the cytolytic activity was determined by radioisotope release into culture supernatant. Data are representative of two separate experiments. 6090 CTL RESPONSE DURING E. cuniculi INFECTION

FIGURE 5. CD8ϩ T cells from E. cuniculi- infected animals are cytolytic toward infected targets. Female C57BL/6 mice of 5–8 wk of age were infected with 1 ϫ 107 spores of E. cuniculi as described in Materials and Meth- ods. Spleen cells from the infected animals (three per group) were pooled, and CD8ϩ T cells were isolated by magnetic separation. The total spleen cell population (TC) as well as the CD8-enriched (CD8ϩ) and -depleted (CD8Ϫ/Ϫ) fractions were collected and cul- tured in the presence of irradiated spores and feeder cells. After a 5-day incubation, viable cells were incubated with 51Cr-labeled unin- fected or infected macrophages at various E:T ratios. After a 4-h incubation, cytolytic activ- ity was measured and calculated as described above. animals were collected; CD8ϩ T cells were isolated by magnetic the infected animals showed disseminated infection in the liver and separation. CD8ϩ T cell-enriched and -depleted fractions were col- spleen. In contrast, very few parasites were observed in the organs lected and cultured in vitro in the presence of irradiated parasites of infected parental wt mice. Similar to parental C57BL/6 mice, and feeder cells. Nondepleted, whole spleen cells were cultured in CD4Ϫ/Ϫ animals were able to resolve E. cuniculi infection. These the presence of Ag alone. After 5 days, the cytolytic effect of total findings suggest that CD8ϩ T cell priming during E. cuniculi in- ϩ ϩ splenocytes and the CD8 T cell-enriched and CD8 T cell-de- fection may be independent of CD4ϩ T cells. The role of CD4ϩ T ϩ pleted populations was evaluated. Purified CD8 T cells exhibited cells in the induction of the CD8ϩ T cell response against viral increased CTL activity compared with the total splenocyte popu- infections has been described previously (34). Infection with lym- lation at all E:T ratios (Fig. 5). On the contrary, the spleen cell phocytic choriomeningitis virus in CD4Ϫ/Ϫ animals results in re- ϩ population depleted of CD8 T cells failed to show any significant duced CD8ϩ T cell immunity and viral clearance (35). Conversely, ϩ cytolysis of infected targets. Target cell lysis with the CD8 T cell a lack of CD4ϩ T cells does not affect the CD8ϩ T cell response depleted population was similar to the uninfected controls. in mice during infection with vaccinia virus (36). It is very likely that under certain circumstances, depending upon the type of in- Discussion fection, CD8ϩ T cells in the absence of CD4ϩ T cells are primed E. cuniculi previously observed in laboratory animals has been through alternate redundant mechanisms. Such mechanisms have been reported to exist in some microbial infections (37, 38). The reported in immunocompromised patients (5). Recent studies have ϩ ϩ increasingly recognized E. cuniculi as an opportunistic infection in precise role of CD4 T cells in the induction of a CD8 T cell response during E. cuniculi infection is being further evaluated. individuals with AIDS (10, 11). E. cuniculi also shares biological ϩ features with other microsporidian that are responsible for mor- CD8 T cell-mediated protection has been shown to occur via bidity and mortality in HIV-infected patients (2). a cytolytic effect on the infected cells, via cytokine production, or The immune response generated during natural E. cuniculi in- both. During infection with lymphocytic choriomeningitis virus, fection has not been well studied. Current literature emphasizes an mice lacking the PF gene are unable to clear the virus due to a lack ϩ ϩ important role for T cells in protective immunity (18). Although a of cytotoxic activity of CD8 T cells (39). In contrast, CD8 - cellular immune response is reported to be essential for immunity dependent immunity against vaccinia virus is largely mediated by ϩ against the parasite, the role of different T cell subtypes in protec- IFN-␥ production (40). IFN-␥-secreting CD8 T cells are believed tion against E. cuniculi infection is not well understood. In the to be important for impeding the chronic Toxoplasma infection ϩ present study, we show that CD8ϩ T cells play an important and (41). CD8 CTLs from Toxoplasma gondii-infected animals ex- perhaps essential role in immunity against this parasite. The pro- hibit in vitro cytolytic activity against syngeneic targets (33). How- tection was shown to be dependent upon a lytic effect of these cells ever, a lack of cytolytic capability in the PF-deficient animals did on the infected targets, as mice deficient in the PF gene succumbed not significantly alter their protective immunity (42). Similarly, ϩ to infection almost at the same time as CD8Ϫ/Ϫ mice. It is possible although CD8 T cells are crucial for resistance against rodent that in addition to CD8ϩ T cells, NK cells that are increased during malaria (43), the protective immunity is independent of PF-medi- early infection and are known to be abundant in PF (30) also play ated cytotoxicity (44). The splenocytes from E. cuniculi-chal- a role in protective immunity against E. cuniculi. However, a total lenged animals were found to exhibit significant lytic activity ϩ splenocyte population that has been depleted of CD8ϩ T cells is against the infected cells. The CD8 CTLs generated during the unable to lyse the parasite-infected targets. Moreover, the suscep- infection mediated the lysis of infected targets. However, unlike T. tibility of CD8Ϫ/Ϫ and SCID mice to E. cuniculi infection rules out gondii and malarial infection, lack of a PF gene compromised the any major role for these cells in the outcome of infection. ability of these mice to withstand E. cuniculi infection. Similar to ϩ Ϫ Ϫ CD8ϩ T cells are known to play an important role in a wide mice lacking CD8 T cells, tissues from PF / mice showed variety of intracellular infections. These cells have been reported greater parasite multiplication. to be critical for many viral, bacterial, and parasitic infections (21, Our data emphasize a critical role for CD8ϩ CTLs during E. 31–33). Our studies demonstrate an early activation and substantial cuniculi infection. Based on our findings, we postulate the follow- increase in the CD8ϩ T cell population during E. cuniculi infec- ing: E. cuniculi infection results in early activation and prolifera- tion. Gene KO mice deficient in CD8ϩ T cells were highly sus- tion of CD8ϩ CTLs. 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