Adoptive Transfer of CD8α+ Dendritic Cells (DC) Isolated from Mice Infected with muridarum Are More Potent in Inducing Protective Immunity Than CD8 α− This information is current as DC of October 1, 2021. Laura Bilenki, Shuhe Wang, Jie Yang, Yijun Fan, Lei Jiao, Antony George Joyee, Xiaobing Han and Xi Yang J Immunol 2006; 177:7067-7075; ;

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Adoptive Transfer of CD8␣؉ Dendritic Cells (DC) Isolated from Mice Infected with Chlamydia muridarum Are More Potent in Inducing Protective Immunity Than CD8␣؊ DC

Laura Bilenki, Shuhe Wang, Jie Yang, Yijun Fan, Lei Jiao, Antony George Joyee, Xiaobing Han, and Xi Yang1

Chlamydial infections are serious public health concerns worldwide. In this study, we examined the role of dendritic cell (DC) subsets in inducing protective immunity against chlamydial infection using an adoptive transfer approach. We found that CD11c؉CD8␣؉ double-positive, DP) DC, compared with CD11c؉CD8␣؊ (single-positive, SP) DC isolated from infected mice, are more potent inducers) of protective immunity. Specifically, mice pretreated with DPDC from infected mice, upon infection with mouse pneumonitis (MoPn), experienced significantly less severe body weight loss and in vivo chlamydial growth. Analysis of MoPn-driven cytokine production by immune cells revealed that mice that were treated with DPDC produced significantly higher levels of Th1 Downloaded from (TNF-␣, IFN-␥, and IL-12) but lower levels of Th2 (IL-4, IL-5, and IL-13)-related cytokines than the recipients of SPDC following infection challenge. Moreover, DPDC-treated mice displayed significantly higher levels of MoPn-specific IgG2a production and delayed- type hypersensitivity responses compared with SPDC-treated mice. Furthermore, DPDC isolated from infected mice produced higher amounts of IL-12 and IL-10 in vitro in comparison with SPDC. These data indicate that CD8␣؉ DC have a significantly higher capacity in inducing protective immunity compared with CD8␣؊ DC, demonstrating the crucial role of DC1-like cells in eliciting protection against C. trachomatis infection The Journal of Immunology, 2006, 177: 7067–7075. http://www.jimmunol.org/

hlamydiae are globally important bacterial pathogens mouse models of respiratory and genital tract infections (10). It has that cause variety of human infectious diseases, including been demonstrated that the pattern of cytokine responses is im- C pneumonia, trachoma, conjunctivitis, and sexually trans- portant in the regulation of immune responses to Chlamydia (11, mitted diseases (1–4). There are two chlamydial that cause 12), and T cell-mediated immunity is the major protective mech- human diseases. Chlamydia trachomatis causes infant pneumonia, anism against chlamydial infection (13–23). It was reported that trachoma, and sexually transmitted diseases, including pelvic in- trachoma patients with severe conjunctival scarring show impaired ␥

flammatory diseases (2, 4). Chlamydia pneumoniae cause pneu- cell-mediated immune responses and low IFN- production with by guest on October 1, 2021 monia, bronchitis, and, more recently, was found to be associated high levels of IgG Ab production (24–26). We previously found with cardiovascular diseases, especially atherosclerosis (5, 6) and, that the mouse strain that produces higher organism-driven IFN-␥ in some degree, with neurological diseases such as Alzheimer’s production shows significantly more resistance to chlamydial in- disease (7, 8). There is no vaccine for chlamydial diseases at the fection (10, 17) and that IFN-␥ gene knockout (KO) mice, IL-12 present time. An urgent prerequisite for the rational development KO mice, and IL-18 KO mice exhibit much more serious diseases of effective and safe prophylactic approaches to chlamydial dis- when compared with wild-type mice (27, 28). Studies by other eases is a better understanding of the mechanisms for protective groups also demonstrated the extreme importance of IFN-␥ and immunity operative during the infection. Th1 immune responses in host defense against chlamydial infec- Although the key mechanism underlying the protective re- tion (14, 22, 23, 25). sponses to Chlamydia is yet to be addressed, significant advances Dendritic cells (DC) have been shown to be the most efficient have been made in recent years largely due to the progress in APC in priming T cells. Based on functional preferences in di- experimental studies using murine models and epidemiological recting Th1 or Th2 differentiation, APC have been divided recently studies in humans (9, 10). Chlamydia muridarum, formally called into type 1 or type 2 APC by some investigators (29–31). Some C. trachomatis mouse pneumonitis (MoPn),2 is commonly used in lineage and phenotypic differences have been identified between the different types of DC. It has been reported in human studies that monocyte-derived CD11cϩ DC (DC1) polarize naive T cells Ϫ Laboratory for Infection and Immunity, Departments of Immunology and Medical toward Th1-like, while CD11c DC (DC2) directs T cells to Th2- Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, like, cells (32, 33). Mouse DCs normally express CD11c and can Canada be divided into CD8␣ϩ and CD8␣Ϫ subsets (34). Some studies Received for publication April 18, 2006. Accepted for publication August 16, 2006. found that CD8␣ϩ lymphoid DC (DC1) primes naive CD4 T cells The costs of publication of this article were defrayed in part by the payment of page to Th1, whereas CD8␣Ϫ myeloid DC (DC2) primes CD4 T cells charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. toward Th2 differentiation (35, 36). 1 Address correspondence and reprint requests to Dr. Xi Yang, Laboratory for Infec- The capability of DC in presenting chlamydial Ags has been tion and Immunity, Department of Medical Microbiology, Faculty of Medicine, Uni- demonstrated by in vitro and in vivo studies (37, 38). Su et al. (37) versity of Manitoba, Room 523, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada. E-mail address: [email protected] 2 Abbreviations used in this paper MoPn, Chlamydia trachomatis mouse pneumonitis IFU, inclusion-forming unit; iSPDC, infected SPDC; KO, knockout; MOMP, major strain; DC, dendritic cell; DPDC, CD11cϩCD8␣ϩ DC; SPDC, CD11cϩCD8␣Ϫ DC; outer membrane protein; nDPDC, naive DPDC; nSPDC, naive SPDC; SPG, sucrose- EB, elementary body; DTH, delayed-type hypersensitivity; iDPDC, infected DPDC; phosphate-glutamic acid.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 7068 DC SUBSETS IN CHLAMYDIAL INFECTION showed that vaccination with murine bone marrow-derived DC (26). Briefly, spleen and lymph node cells were cultured at a concentration pulsed with Chlamydia induced a protective immune response in a of 7.5 ϫ 106 and 5.0 ϫ 106 cells/ml, respectively, alone or with heat- ϫ 5 genital infection model. Similar results were obtained in a study inactivated MoPn (1 10 IFU/ml) at 37°C in complete culture medium: RPMI 1640 containing 10% heat-inactivated FBS, 25 ␮g/ml gentamicin, 2 Ϫ5 using cultured DC line (D3SC/1) (39). Lu and Zhong (40) reported mM L-glutamine, and 5 ϫ 10 2-ME (Kodak). Duplicate cultures were that IL-12 production is required for chlamydial Ag-pulsed DC to established from the spleen and lymph node cells of individual mice in induce protection against infection. We also reported that MoPn each group. Culture supernatants were harvested at 72 h for the measure- infection in the peritoneum induced the maturation of DC (41) and ment of cytokines by ELISA using purified (capture) and biotinylated (de- tection) Abs as described previously (17, 27). Abs purchased from BD DC from Chlamydia-infected C57BL/6 mice express higher levels Pharmingen were used for ELISA measuring IL-4, IL-5, IL-10, IL-12, of IL-12 than those from naive mice (42, 43). However, a critical IFN-␥, and TNF-␣. IL-13 was determined using paired Abs purchased analysis of the DC subsets relating to differential host responses to from R&D Systems. Chlamydia has yet to be performed. Determination of delayed-type hypersensitivity (DTH) responses In the present study, we demonstrated an expansion of CD8␣ϩ DC population in the mice infected with C. muridarum. The com- MoPn-specific DTH was measured as described previously (17). Mice ␣ϩ ␣Ϫ were injected in the hind footpads with 25 ␮l of heat-inactivated MoPn EB parison of the ability of sorted CD8 and CD8 DC subsets in (5 ϫ 104 IFU) in one side and the same volume of SPG buffer in the other transferring protection against C. muridarum infection challenge side. The difference in thickness between the two footpads at 24, 48, and ϩ Ϫ showed that CD8␣ subset was more protective than CD8␣ sub- 72 h was used as a measure of the DTH response. No measurable difference set. Additionally, we have analyzed cytokine patterns of DC sub- was found between the two footpads injected with heat-inactivated EB or sets and the immune responses generated in the recipients of dif- SPG buffer in uninfected control mice. ferent DC subsets to elucidate potential mechanisms involved in Determination of MoPn-specific Ab levels Downloaded from protection against chlamydial infection induced by transferred DC Chlamydia-specific murine IgE were measured by ELISA using Abs pur- subsets. chased from BD Pharmingen. For the measurement of MoPn-specific IgE, serum samples were pretreated by incubating twice with 50% slurry of Materials and Methods protein G-Sepharose (Pharmacia Biotech) to remove IgG (44). This treat- Mice ment removes Ͼ95% IgG from the sera. For MoPn-specific IgG1 and IgG2a, sera without protein G-Sepharose treatment were tested. The Abs C57BL/6 mice (7–10 wk old) used in the study were bred at the University for IgG1 and IgG2a ELISA were purchased from Southern Biotechnology of Manitoba breeding facility. Animals were used in accordance with the Associates. Briefly, to determine MoPn-specific serum Abs, ELISA plates http://www.jimmunol.org/ guidelines issued by the Canadian Council on Animal Care. were coated overnight with MoPn EB in bicarbonate buffer (0.05 M (pH 9.6)). After blocking for 90 min with a 2% BSA and 0.05% Tween 20 solution and extensive washing, serially diluted sera were incubated for 2 h The culture and preparation of C. muridarum (MoPn) were performed as at 37°C. The plates were washed, and biotinylated goat anti-mouse Ab described previously (17, 27). Briefly, MoPn was cultured in HeLa 229 was added and incubated for overnight at 4°C. Alkaline phosphatase- cells in Eagle’s MEM containing 10% FBS and 2 mM L-glutamine for 48 h. conjugated streptavidin (Jackson ImmunoResearch Laboratories) was For inoculum preparation, infected cells were harvested with sterile glass added, and the plates were kept for incubation at room temperature for 45 beads and partially purified by successive 15-min 500 ϫ g and 30-min min. After extensive washing of the plates, p-nitrophenyl phosphate (in 0.5 30,000 ϫ g centrifugations. The partially purified organisms were resus- mM MgCl2 and 10% diethanolamine (pH 9.8)) was added, and the reaction pended in sucrose-phosphate-glutamic acid (SPG) buffer, and frozen at was allowed to proceed to 60 min. The plates were read with a microplate by guest on October 1, 2021 Ϫ80°C until used. The same seed stock of MoPn was used throughout the reader (Versamax; Molecular Devices) at 405 nm. study. For the anti-Chlamydia Ab ELISA, the MoPn elementary body (EB) Fluorescence activated cell sorting and adoptive transfer of DC preparations were further purified by step gradient centrifugation using 35% Renografin (Squibb). Spleens from infected or naive mice were aseptically removed, and DC were isolated using MACS (Miltenyi Biotec) CD11c columns according to Infection of mice and quantification of chlamydial in vivo the manufacturer’s instructions. Briefly, spleens were digested with Col- growth lagenase D (Boehringer Mannheim). Single-cell suspensions were prepared in PBS with 0.5% BSA. CD11c microbeads were added to the cell sus- ϫ 3 Mice were inoculated intranasally with 1–3 10 inclusion-forming units pensions and were isolated using MACS LS columns. Purity of isolated (IFU) MoPn. Body weights of the mice before (day 0) and after inoculation DC was between 94 and 99%. Freshly isolated DCs from naive or with MoPn were recorded daily. The mice were killed at specified time, infected mice were costained with PE anti-mouse CD11c (clone HL3, and the lungs were aseptically isolated and homogenized using a cell Armenian IgG1, ␭) and FITC anti-mouse CD8␣ (clone 53-6.7, rat grinder in SPG buffer. Tissue suspensions were spun down at 1900 ϫ g for ␬ ϩ ␣ϩ Ϫ (LOU/Ws1/M) IgG2a, ) and sorted for CD11c CD8 (double-positive, DP) 30 min at 4°C to remove coarse tissues and debris and frozen at 80°C DC (DPDC) or CD11cϩCD8␣Ϫ (single-positive, SP) DC (SPDC) using until being tested. For MoPn quantitation, HeLa 229 cells were grown to EPICS ALTRA flow cytometer (Beckman Coulter). Sorted DC subsets were confluence in 96-well flat-bottom microtiter plates and washed in 100 ␮lof ␮ washed once with PBS and were adoptively transferred to syngeneic naive HBSS. The monolayers were then inoculated in triplicate with 100 lof mice through i.v. injection at 0.5 ϫ 106 sorted DC/mouse. To examine the serially diluted organ tissue supernatants from mice infected with MoPn. ␮ surface marker expression, the sorted DPDC and SPDC were analyzed for After2hofincubation at 37°C, plates were washed,and 200 lofMEM MHC class II (FITC anti-mouse I-Ab (clone 26-9-17, mouse (C3H) IgG2a,␬) containing cycloheximide (1.5 ␮g/ml), gentamicin (20 ␮g/ml), and van- ␬ ␮ and CD80 (PE anti-mouseCD80 (clone 16-10A1, Armenian hamster IgG2, ). comycin (25 g/ml) was added to each well. The plates were incubated for Fluorescence-conjugated mAbs with appropriate isotype controls were pur- 48 h at 37°C in 5% CO2. Following the incubation, the culture medium was chased from BD Pharmingen. removed, and the cell monolayers were fixed with absolute methanol. To identify chlamydial inclusions, plates were incubated with a Chlamydia Statistical analysis genus-specific murine mAb and stained with goat anti-mouse IgG conju- gated to HRP and developed with substrate (4-chloro-1-napthol; Sigma- Ab titers (ELISA) were converted to logarithmic values and analyzed using Aldrich). The number of inclusions was counted under a microscope at the unpaired Student’s t test. IFU detection and cytokine production levels ϫ200 magnification. Five fields through the midline of each well were were analyzed using the unpaired Student’s t test. counted. The chlamydial levels in each organ were calculated based on dilution titers of the original inoculum. Results MoPn infection induces expansion of CD8␣ϩ DC population, Spleen and lymph node cell cultures and cytokine determination which shows different pattern of surface marker expression Mice were killed at day 12 following intranasal inoculation with MoPn and and cytokine production the spleens and draining (mediastinum) lymph nodes were aseptically re- moved. The cytokine production by spleen and lymph node cells was ex- We first examined the effect of MoPn infection on DC population amined. Single-cell suspensions were cultured as described previously in vivo. As shown in Fig. 1A, DC isolated from MoPn-infected The Journal of Immunology 7069

FIGURE 1. MoPn infection induces expansion of CD8␣ϩ DCs, which ex- press higher level costimulatory mole- cules and produce significantly higher levels of IL-12 and IL-10 compared Downloaded from with CD8␣Ϫ DCs. Mice were infected intranasally with MoPn (1000 IFU) and were sacrificed 7 days postinfection. A, Spleen cells were isolated and CD11cϩ cells were purified using MACS CD11cϩ cell isolation column. Purified CD11cϩ cells were analyzed by flow http://www.jimmunol.org/ cytometry for CD8␣ϩ expression. CD11cϩ cells from naive mice were used as controls. B, Purified CD11cϩ spleen cells from MoPn-infected and uninfected mice were double stained for CD8␣ and CD80 or CD86 mole- cules using conjugated Abs and ana- lyzed by flow cytometry. C, Spleen

cells from MoPn-infected and unin- by guest on October 1, 2021 fected mice were stained for surface CD11c and CD8␣ and subjected to cell sorting resulting in CD11cϩCD8␣ϩ and CD11cϩCD8␣Ϫ subpopulations. The DC subsets were placed in culture for 72 h, and cytokines in the superna- p Ͻ ,ء .tants were measured by ELISA p Ͻ 0.001. One representative ,ءء ;0.05 experiment of three independent exper- iments is shown.

mice showed significant increase of CD8␣ϩ population compared proportion of DPDC expressed CD80 (57.9% (34.2/(34.2 ϩ 24.9)) with those from naive mice (54.2 vs 21.1%). We then analyzed the vs 37% (15.4/(15.4 ϩ 25.5))) and CD86 (60.6% (36.1/(36.1 ϩ costimulatory surface markers expression of DPDC and SPDC 23.5)) vs 36.6% (14.4/(14.4 ϩ 26.0))) molecules on their cell sur- subsets. As shown in Fig. 1B, compared with SPDC, a higher face. Similar differences in CD80 (43.5 vs 38.5%) and CD86 (30.1 7070 DC SUBSETS IN CHLAMYDIAL INFECTION

Adoptive transfer of CD11cϩCD8␣ϩ DCs from infected mice reduces body weight loss and chlamydial in vivo growth To compare the effectiveness of DC subsets in inducing protection against chlamydial infection, naive C57BL/6 mice were adoptively transferred i.v. with DPDC or SPDC isolated from syngeneic MoPn-infected or naive mice followed by intranasal infection with MoPn. As shown in Fig. 2, the body weight loss was significantly less in mice given DP DC from infected DPDC (iDPDC) mice following MoPn infection challenge than mice without DC adop- tive transfer. The overall physical condition of the mice that re- ceived iDPDC was also better (i.e., more activity, less fur ruffling and dehydration). The recipients of SP DC from infected SPDC (iSPDC) mice also showed less body weight loss than the control mice, but still more than iDPDC recipients. In contrast, the adop- tive transfer of DC subsets from naive mice (CD11cϩCD8␣ϩ (nDPDC) or CD11cϩCD8␣Ϫ (nSPDC)) had no significant effect FIGURE 2. Less body weight loss following chlamydial infection in mice administered with CD8␣ϩ DC and exposed to MoPn infection. Naive recipient mice (C57BL/6) were delivered CD8␣ϩ or CD8␣Ϫ DC isolated from the spleen of MoPn-infected (intranasal, 1000 IFU) mice or naive Downloaded from C57BL/6 mice followed by intranasal challenge infection with same chla- mydial strain, MoPn (3000 IFU). Mice were monitored daily for body weight changes. Each point represents the mean Ϯ SD of four mice. The p Ͻ ,ء .original body weights of the mice were similar between the groups 0.05, compared with mice without cell transfer. One representative exper- iment of three independent experiments with similar results is shown. http://www.jimmunol.org/ vs 9.2%) expression between DPDC and SPDC were found in uninfected mice, although the levels were lower than correspond- ing subsets from infected mice. To test the cytokine production pattern of the DC subsets, sorted DPDC and SPDC were placed in culture, and the spontaneous cytokine production was measured. As shown in Fig. 1C, DPDC produced significantly higher levels of IL-12 and IL-10 than SPDC. Similar differences were found by guest on October 1, 2021 between the subsets of DCs from uninfected mice, although the absolute levels were lower than those in infected mice. These re- sults indicate that CD8␣ϩ DC from infected mice express higher level of costimulatory molecules and produce higher immune reg- ulatory cytokines than CD8Ϫ DC.

FIGURE 4. Mice treated with CD8␣ϩ DC and infected with MoPn ex- hibit less inflammatory responses in the lung. The lungs from mice treated with CD8␣ϩ or CD8␣Ϫ DC subsets and intranasally (i.n.) infected with FIGURE 3. Less chlamydial growth in vivo following MoPn infection MoPn at day 12 postinfection were analyzed for histological changes by in mice treated with CD8␣ϩ DC isolated from Chlamydia-infected mice. H&E staining. Graphs shown are a magnification of ϫ200 with photo The different groups of mice shown in Fig. 2 were sacrificed on day 12 insets shown at a magnification of ϫ400. A, Mice treated with postinfection, and the different organs (lung, liver, and heart) were ana- CD11cϩCD8␣ϩ DC from infected mice and infected i.n. with MoPn. B, lyzed for in vivo chlamydial growth as described in Materials and Meth- Mice treated with CD11cϩCD8␣Ϫ DC from infected mice i.n. and infected ods. One representative experiment of three independent experiments with with MoPn. C, Mice were infected with MoPn without prior DC adoptive represents p Ͻ 0.05, compared with mice with- transfer. One representative experiment of three independent experiments ء .similar results is shown out cell transfer. is shown. The Journal of Immunology 7071

immune response, we tested DTH and Ab responses in these mice following MoPn infection. As shown in Fig. 5, while DTH response was observed in all groups of mice, the recipients of CD8␣ϩ DC from iDPDC mice displayed markedly stronger DTH reaction than the other groups of mice. The data suggest that CD8␣ϩ DC from infected mice are more powerful in inducing cell-mediated immune responses in vivo during chlamydial infection. Ab measurement showed that the levels of MoPn-specific IgG2a were significantly higher in mice treated with CD8␣ϩ DC from iDPDC mice compared with those treated with PBS (Fig. 6). The transfer of CD8␣Ϫ DC from iSPDC mice or DC subsets from naive mice had no significant effect on IgG2a production in the recipient mice. In addition, the levels of MoPn-specific IgG1 were significantly lower in the mice pretreated with DPDC or SPDC from infected mice than the control mice, with most obvious re- duction in iDPDC recipients (Fig. 6). Taken together, the results FIGURE 5. Significantly stronger DTH responses in mice treated with indicate that the type of immune responses, including DTH and the ␣ϩ CD8 DC and infected with MoPn. For DTH response, mice (four mice isotypes of Ab production, were altered in mice pretreated with per group) were treated and infected as described in Fig. 2. At 11 days DCs isolated from MoPn-infected mice following challenge infec- postintranasal challenge infection, the footpads of different groups of mice Downloaded from were challenged with heat-inactivated MoPn (left footpad) or SPG (right tion. In particular, adoptive transfer of iDPDC increased DTH but footpad), and the thicknesses of the footpads were measured at 24, 48, and decreased IgG1 and IgE Ab responses. 72 h following dead MoPn injection. The difference in the thickness of ϩ footpads with or without MoPn injection was used as a measure of DTH Mice pretreated with CD8␣ DC isolated from MoPn-infected responses as described in Material and Methods. Data show the mean Ϯ mice exhibit increased MoPn-driven Th1, but reduced Th2, represents p Ͻ 0.05, compared with mice without DC adoptive cytokine production upon chlamydial infection ء .SD transfer. One of three independent experiments with similar results is http://www.jimmunol.org/ shown. To examine the mechanisms involved in protection induced by DC subsets in the recipient mice following infection challenge, we investigated the organism-driven cytokine production by spleen on mice conditions. The analysis of chlamydial loads in the lung and draining lymph node cells from different groups of mice. The showed that the recipients of iDPDC had significantly lower results revealed that both spleen and lymph node cells of MoPn- (1000-fold less) in vivo chlamydial growth than the control mice infected mice pretreated with iDPDC produced significantly higher ␣ ␥ treated with PBS only (Fig. 3). In correlation with the finding in levels of TNF- , IFN- , and IL-12 compared with the mice with- body weight changes, the recipients of iSPDC also showed less out DC adoptive transfer (Fig. 7). Notably, the recipients of iSPDC chlamydial growth in the lung than control mice, but their chla- also showed increased production of the Th1-related cytokines but by guest on October 1, 2021 mydial loads were still 100 times higher than the iDPDC recipients not to the degree in the iDPDC recipients. Interestingly, the recip- ␣ (Fig. 3). In contrast, the adoptive transfer of nDPDC or nSPDC ients of nDPDC also showed significant increase of TNF- and ␥ failed to show protective effects (Figs. 2 and 3). In addition, the IFN- , although they were lower than that in the recipients of DC recipients of iDPDC showed most mild pathological changes in the subsets from infected mice. In contrast, Th2-like cytokines such as lung compared with other groups of mice, as demonstrated by less IL-4, IL-5, and IL-13 were significantly reduced in iDPDC-treated cellular infiltration (Fig. 4). The results indicate that the adoptive mice (Fig. 8). The transfer of iSPDC also appeared to decrease Th2 transfer of either DC subsets from infected mice can induce sig- cytokine production but to a lesser degree. These results, together ␣ϩ with the finding in the types of immune responses (DTH and Ab nificant protective immunity in the recipients, but that CD8 DC ϩ Ϫ ␣ are much more potent in inducing protection than CD8␣ DC. isotypes), demonstrate a powerful role of CD8 DC in promoting protective Th1-type immune responses to chlamydial infection. CD8␣ϩ DC induce stronger DTH reaction and MoPn-specific IgG2a Ab protection than CD8␣-DC Discussion To examine the relationship between the degree of protection ob- In the present model, we have shown that the transfer of CD8␣ϩ served in mice delivered with different DC subsets and the types of DC, compared with CD8␣Ϫ DC, isolated from C. trachomatis

FIGURE 6. Recipients of DC from in- fected mice produce significantly lower lev- els of serum IgG1 following challenge infec- tion. The sera collected from mice (four mice per group) post adoptive transfer of DC and postintranasal (day 12) MoPn infection as described in Fig. 2 were determined for MoPn-specific IgG1 and IgG2a using ELISA. Data are presented as mean Ϯ SD. p Ͻ 0.001, compared with ,ءء ;p Ͻ 0.05 ,ء mice without DC adoptive transfer. One rep- resentative experiment of three independent experiments is shown. 7072 DC SUBSETS IN CHLAMYDIAL INFECTION

FIGURE 7. Recipients of DC from infected mice produce significantly higher MoPn-driven Th1 cy- tokines following challenge infection. Mice (four mice/group) were treated and infected as described in the legend to Fig. 2, and were sacrificed at 12 days postinfection. Spleen and lymph node cells were cultured with UV-inactivated MoPn as de- scribed in Materials and Methods. TNF-␣, IFN-␥ and IL-12 in 72 h culture supernatants were deter- mined by ELISA. Data are presented as the mean Ϯ Downloaded from -p Ͻ 0.001, com ,ءء ;p Ͻ 0.05 ,ء .SD of each group pared with mice without DC adoptive transfer. One representative experiment of three independent ex- periments is shown. http://www.jimmunol.org/ by guest on October 1, 2021

MoPn-infected mice are much more capable of transferring pro- different DC subsets (45, 46). These subsets were further examined tective immunity to naive mice to subsequent challenge infection. by performing coculture with T cells or adoptively transferred to To our knowledge, this is the first report showing variable capacity recipients after pulsing with Ags (45). A few studies used splenic of DC subsets in inducing protective immunity against chlamydial CD8␣ϩ and CD8␣Ϫ DC subsets, in which the DC subsets were infection. Moreover, our results showed that the DC subsets were pulsed with Ags in vitro followed by adoptive transfer of the cells different in costimulatory molecule expression and cytokine pro- (46). Although these approaches are useful in testing the immu- duction. In particular, CD8␣ϩ DC from infected mice secreted nological function of various Ags, the handling of these Ags by IL-12 and IL-10 at significantly higher levels compared with DC subsets in vitro may not necessarily reflect the physiological CD8␣Ϫ DC from the same mice or either subset isolated from process in vivo. In contrast, in the present study, the Ag-processing naive mice. The ability of CD8␣ϩ DC to produce higher levels of steps and the development of DC subsets occurred in vivo condi- these cytokines indicates a pertinent role for these cytokines in the tions following infection. Furthermore, these DC subsets were transfer of protective immunity against subsequent challenge adoptively transferred to recipient mice immediately after isolation infection. without any further culture or manipulation; these results thus The approach used in this study is more physiological than most arguably more mimicking the function of DC subsets in vivo. In previously reported studies on the function of DC subsets. Al- fact, the conclusion generated from the current approach and other though there is no direct evidence showing the carrying of chla- studies (36) is very consistent in that CD8␣ϩ DC are more potent mydial Ags by the transferred DC, the fact that the sera collected in inducing Th1-like responses than CD8␣Ϫ DC. The powerful from recipients of DC from infected mice without further chal- Th1-inducing function of CD8␣ϩ DC is further supported by the lenge infection showed Ab to MoPn Ags by Western blot analysis finding that adoptive transfer of CD8␣ϩ DC from naive mice also (data not shown) indicated that the DC from infected mice carry increased IFN-␥ and TNF-␣ production (Fig. 7), although this was chlamydial Ags and/or antigenic peptides. Since the transferred not strong enough to alter the type of immune responses (Figs. 5 DC subsets from infected mice carried chlamydial Ags/peptides, a and 6) and the outcome of protection (Figs. 2 and 3). Since mice faster protective immunity can develop. At the same time, the dif- show significant increase of CD8␣ϩ DC following MoPn infection ference in protection between CD8␣ϩ and CD8␣Ϫ subsets from (Fig. 1A), one might argue the mice should be protected even with- infected mice may reflect their difference of potency in inducing out DC transfer. Indeed, we have reported previously that mice protective responses. Notably, most of the previous studies exam- started to recover after MoPn infection at 13–15 days following ined bone marrow-derived or peripheral monocyte-derived DC, intranasal infection even in the absence of DC transfer (17, 47). A which were manipulated in in vitro culture conditions to generate major reason for the natural recovery of the mice is likely because The Journal of Immunology 7073

FIGURE 8. Recipients of DC from in- fected mice produce significantly lower MoPn-driven Th2 cytokines following chal- lenge infection. Spleen and lymph node cell culture supernatants from experiments de- scribed in Fig. 7 were determined for Th2 cytokines (IL-4, IL-5, and IL-13) by ELISA. Downloaded from Data are presented as the mean Ϯ SD of -p Ͻ 0.001, com ,ءء ;p Ͻ 0.05 ,ء .each group pared with mice without DC adoptive trans- fer. One representative experiment of three independent experiments is shown. http://www.jimmunol.org/ by guest on October 1, 2021

of the significant increase of CD8␣ϩ DCs and the subsequent de- Su et al. (37) and Shaw et al. (38) have reported that adoptive velopment of protective adaptive immune responses. transfer of whole Chlamydia organism-pulsed bone marrow- ϩ Notably, although our data strongly suggest that CD8␣ DCs derived DC induce strong Th1 responses and protection to chal- are DC1-like cells, which preferentially induce Th1-like responses, lenge infection, while major outer membrane protein (MOMP)- Ϫ the CD8␣ DCs in our model are not typical DC2-like cells. In pulsed bone marrow-derived DC induce Th2-type responses and Ϫ fact, the adoptive transfer of CD8␣ DCs from infected mice also failed to provide protection (48). In addition, these authors also enhanced Th1-related cytokine production (Fig. 7) and inhibited found that the DC pulsed with whole chlamydial organism produce Th2-related cytokine production (Fig. 8) and generated certain de- higher IL-12 than the DC pulsed with MOMP. Therefore, it is gree of protection (Figs. 2 and 3). One possible reason for this is likely that the two systems preferentially elicit different DC sub- that the CD8␣Ϫ DCs are still a heterogeneous population, which sets, i.e., the former mainly induce DC1-like cells that are func- includes both DC1- and DC2-like cells, and the DC1-like subset is ϩ tionally similar to the CD8␣ DC in the present study, thus in- again predominant. Indeed, we found that within the CD8␣Ϫ DC ducing strong Th1 responses, while the latter prefer the population, further separation based on CD4 expression could ob- ϩ Ϫ development of DC2-like cells. Moreover, although the DC with tain distinct CD4 and CD4 DC subsets, and the former pro- ␣ duced least amount of IL-12 in the in vitro culture compared with CD8 marker may not necessarily be the cell to be focused on for CD8␣ϩCD4Ϫ and CD8␣-CD4Ϫ DC (X. Han and X. Yang, un- future vaccine studies, our present finding suggests that it is very published data). Therefore, using CD8␣ as a sole discriminative important to realize the potential development and involvement of marker for DC1- and DC2-like cells is at least an over simplicity various DC subsets following chlamydial infection or vaccination. of DC biology and function. It is very likely that heterogeneity of A rather surprising but also very interesting finding in the DC in vivo is far beyond our imagination. present study is the higher IL-10 (in addition to higher IL-12) ϩ The finding that different DC subsets induce different levels of production by CD8␣ DCs from infected mice, which confer protection may be helpful in understanding the previous reports on strong protective immunity against chlamydial infection challenge. the role of DC in chlamydial immunobiology and opens the door Although IL-12 has been consistently found to be associated with for further immunological and vaccination studies. For example, protection (28, 40), higher IL-10 production has been previously 7074 DC SUBSETS IN CHLAMYDIAL INFECTION linked to susceptibility to C. trachomatis infection and immuno- DC subset(s) beneficial for preventive and therapeutic strategies pathology in mouse and human chlamydial diseases (17, 47, 49). for chlamydial diseases. It remains unclear why a DC subset, which produces higher levels of these two apparently functionally counteractive cytokines, Disclosures showed dominant protective phenotype. However, there are sev- The authors have no financial conflict of interest. eral possibilities, which may account for this observation. First, it is possible that the relative level of production of these two cyto- References ϩ kines by CD8␣ DCs are variable, leading to a higher IL-12:IL-10 1. Dawson, C. R., B. R. Jones, and M. L. Tarizzo. 1981. Programmes for the pre- ratio, thus enhancing Th1 responses. Indeed, our results showed vention of blindness. 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