Adoptive Transfer of CD8α⁺ Dendritic Cells (DC) Isolated from Mice Infected with Chlamydia muridarum Are More Potent in Inducing Protective Immunity Than CD8α⁻ DC

Chlamydiae are globally important bacterial pathogens that cause variety of human infectious diseases, including pneumonia, trachoma, conjunctivitis, and sexually transmitted diseases (1, 2, 3, 4). There are two chlamydial species that cause human diseases. Chlamydia trachomatis causes infant pneumonia, trachoma, and sexually transmitted diseases, including pelvic inflammatory diseases (2, 4). Chlamydia pneumoniae cause pneumonia, bronchitis, and, more recently, was found to be associated with cardiovascular diseases, especially atherosclerosis (5, 6) and, in some degree, with neurological diseases such as Alzheimer’s disease (7, 8). There is no vaccine for chlamydial diseases at the present time. An urgent prerequisite for the rational development of effective and safe prophylactic approaches to chlamydial diseases is a better understanding of the mechanisms for protective immunity operative during the infection.

Although the key mechanism underlying the protective responses to Chlamydia is yet to be addressed, significant advances have been made in recent years largely due to the progress in experimental studies using murine models and epidemiological studies in humans (9, 10). Chlamydia muridarum, formally called C. trachomatis mouse pneumonitis (MoPn),² is commonly used in mouse models of respiratory and genital tract infections (10). It has been demonstrated that the pattern of cytokine responses is important in the regulation of immune responses to Chlamydia (11, 12), and T cell-mediated immunity is the major protective mechanism against chlamydial infection (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). It was reported that trachoma patients with severe conjunctival scarring show impaired cell-mediated immune responses and low IFN-γ production with high levels of IgG Ab production (24, 25, 26). We previously found that the mouse strain that produces higher organism-driven IFN-γ production shows significantly more resistance to chlamydial infection (10, 17) and that IFN-γ gene knockout (KO) mice, IL-12 KO mice, and IL-18 KO mice exhibit much more serious diseases when compared with wild-type mice (27, 28). Studies by other groups also demonstrated the extreme importance of IFN-γ and Th1 immune responses in host defense against chlamydial infection (14, 22, 23, 25).

Dendritic cells (DC) have been shown to be the most efficient APC in priming T cells. Based on functional preferences in directing Th1 or Th2 differentiation, APC have been divided recently into type 1 or type 2 APC by some investigators (29, 30, 31). Some lineage and phenotypic differences have been identified between the different types of DC. It has been reported in human studies that monocyte-derived CD11c⁺ DC (DC1) polarize naive T cells toward Th1-like, while CD11c⁻ DC (DC2) directs T cells to Th2-like, cells (32, 33). Mouse DCs normally express CD11c and can be divided into CD8α⁺ and CD8α⁻ subsets (34). Some studies found that CD8α⁺ lymphoid DC (DC1) primes naive CD4 T cells to Th1, whereas CD8α⁻ myeloid DC (DC2) primes CD4 T cells toward Th2 differentiation (35, 36).

The capability of DC in presenting chlamydial Ags has been demonstrated by in vitro and in vivo studies (37, 38). Su et al. (37) showed that vaccination with murine bone marrow-derived DC pulsed with Chlamydia induced a protective immune response in a genital infection model. Similar results were obtained in a study using cultured DC line (D3SC/1) (39). Lu and Zhong (40) reported that IL-12 production is required for chlamydial Ag-pulsed DC to induce protection against infection. We also reported that MoPn infection in the peritoneum induced the maturation of DC (41) and DC from Chlamydia-infected C57BL/6 mice express higher levels of IL-12 than those from naive mice (42, 43). However, a critical analysis of the DC subsets relating to differential host responses to Chlamydia has yet to be performed.

In the present study, we demonstrated an expansion of CD8α⁺ DC population in the mice infected with C. muridarum. The comparison of the ability of sorted CD8α⁺ and CD8α⁻ DC subsets in transferring protection against C. muridarum infection challenge showed that CD8α⁺ subset was more protective than CD8α⁻ subset. Additionally, we have analyzed cytokine patterns of DC subsets and the immune responses generated in the recipients of different DC subsets to elucidate potential mechanisms involved in protection against chlamydial infection induced by transferred DC subsets.

C57BL/6 mice (7–10 wk old) used in the study were bred at the University of Manitoba breeding facility. Animals were used in accordance with the guidelines issued by the Canadian Council on Animal Care.

The culture and preparation of C. muridarum (MoPn) were performed as described previously (17, 27). Briefly, MoPn was cultured in HeLa 229 cells in Eagle’s MEM containing 10% FBS and 2 mM l-glutamine for 48 h. For inoculum preparation, infected cells were harvested with sterile glass beads and partially purified by successive 15-min 500 × g and 30-min 30,000 × g centrifugations. The partially purified organisms were resuspended in sucrose-phosphate-glutamic acid (SPG) buffer, and frozen at −80°C until used. The same seed stock of MoPn was used throughout the study. For the anti-Chlamydia Ab ELISA, the MoPn elementary body (EB) preparations were further purified by step gradient centrifugation using 35% Renografin (Squibb).

Mice were inoculated intranasally with 1–3 × 10³ inclusion-forming units (IFU) MoPn. Body weights of the mice before (day 0) and after inoculation with MoPn were recorded daily. The mice were killed at specified time, and the lungs were aseptically isolated and homogenized using a cell grinder in SPG buffer. Tissue suspensions were spun down at 1900 × g for 30 min at 4°C to remove coarse tissues and debris and frozen at −80°C until being tested. For MoPn quantitation, HeLa 229 cells were grown to confluence in 96-well flat-bottom microtiter plates and washed in 100 μl of HBSS. The monolayers were then inoculated in triplicate with 100 μl of serially diluted organ tissue supernatants from mice infected with MoPn. After 2 h of incubation at 37°C, plates were washed,and 200 μl of MEM containing cycloheximide (1.5 μg/ml), gentamicin (20 μg/ml), and vancomycin (25 μg/ml) was added to each well. The plates were incubated for 48 h at 37°C in 5% CO₂. Following the incubation, the culture medium was removed, and the cell monolayers were fixed with absolute methanol. To identify chlamydial inclusions, plates were incubated with a Chlamydia genus-specific murine mAb and stained with goat anti-mouse IgG conjugated to HRP and developed with substrate (4-chloro-1-napthol; Sigma-Aldrich). The number of inclusions was counted under a microscope at ×200 magnification. Five fields through the midline of each well were counted. The chlamydial levels in each organ were calculated based on dilution titers of the original inoculum.

Mice were killed at day 12 following intranasal inoculation with MoPn and the spleens and draining (mediastinum) lymph nodes were aseptically removed. The cytokine production by spleen and lymph node cells was examined. Single-cell suspensions were cultured as described previously (26). Briefly, spleen and lymph node cells were cultured at a concentration of 7.5 × 10⁶ and 5.0 × 10⁶ cells/ml, respectively, alone or with heat-inactivated MoPn (1 × 10⁵ IFU/ml) at 37°C in complete culture medium: RPMI 1640 containing 10% heat-inactivated FBS, 25 μg/ml gentamicin, 2 mM l-glutamine, and 5 × 10⁻⁵ 2-ME (Kodak). Duplicate cultures were established from the spleen and lymph node cells of individual mice in each group. Culture supernatants were harvested at 72 h for the measurement of cytokines by ELISA using purified (capture) and biotinylated (detection) Abs as described previously (17, 27). Abs purchased from BD Pharmingen were used for ELISA measuring IL-4, IL-5, IL-10, IL-12, IFN-γ, and TNF-α. IL-13 was determined using paired Abs purchased from R&D Systems.

MoPn-specific DTH was measured as described previously (17). Mice were injected in the hind footpads with 25 μl of heat-inactivated MoPn EB (5 × 10⁴ IFU) in one side and the same volume of SPG buffer in the other side. The difference in thickness between the two footpads at 24, 48, and 72 h was used as a measure of the DTH response. No measurable difference was found between the two footpads injected with heat-inactivated EB or SPG buffer in uninfected control mice.

Chlamydia-specific murine IgE were measured by ELISA using Abs purchased from BD Pharmingen. For the measurement of MoPn-specific IgE, serum samples were pretreated by incubating twice with 50% slurry of protein G-Sepharose (Pharmacia Biotech) to remove IgG (44). This treatment removes >95% IgG from the sera. For MoPn-specific IgG1 and IgG2a, sera without protein G-Sepharose treatment were tested. The Abs for IgG1 and IgG2a ELISA were purchased from Southern Biotechnology Associates. Briefly, to determine MoPn-specific serum Abs, ELISA plates 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 at 37°C. The plates were washed, and biotinylated goat anti-mouse Ab was added and incubated for overnight at 4°C. Alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories) was added, and the plates were kept for incubation at room temperature for 45 min. After extensive washing of the plates, p-nitrophenyl phosphate (in 0.5 mM MgCl₂ and 10% diethanolamine (pH 9.8)) was added, and the reaction was allowed to proceed to 60 min. The plates were read with a microplate reader (Versamax; Molecular Devices) at 405 nm.

Spleens from infected or naive mice were aseptically removed, and DC were isolated using MACS (Miltenyi Biotec) CD11c columns according to the manufacturer’s instructions. Briefly, spleens were digested with Collagenase D (Boehringer Mannheim). Single-cell suspensions were prepared in PBS with 0.5% BSA. CD11c microbeads were added to the cell suspensions and were isolated using MACS LS columns. Purity of isolated DC was between 94 and 99%. Freshly isolated DCs from naive or infected mice were costained with PE anti-mouse CD11c (clone HL3, Armenian hamster IgG1, λ) and FITC anti-mouse CD8α (clone 53-6.7, rat (LOU/Ws1/M) IgG2a,κ) and sorted for CD11c⁺CD8α⁺ (double-positive, DP) DC (DPDC) or CD11c⁺CD8α⁻ (single-positive, SP) DC (SPDC) using EPICS ALTRA flow cytometer (Beckman Coulter). Sorted DC subsets were washed once with PBS and were adoptively transferred to syngeneic naive mice through i.v. injection at 0.5 × 10⁶ sorted DC/mouse. To examine the surface marker expression, the sorted DPDC and SPDC were analyzed for MHC class II (FITC anti-mouse I-Ab (clone 26-9-17, mouse (C3H) IgG2a,κ) and CD80 (PE anti-mouseCD80 (clone 16-10A1, Armenian hamster IgG2,κ). Fluorescence-conjugated mAbs with appropriate isotype controls were purchased from BD Pharmingen.

Ab titers (ELISA) were converted to logarithmic values and analyzed using the unpaired Student’s t test. IFU detection and cytokine production levels were analyzed using the unpaired Student’s t test.

We first examined the effect of MoPn infection on DC population in vivo. As shown in Fig. 1,A, DC isolated from MoPn-infected mice showed significant increase of CD8α⁺ population compared with those from naive mice (54.2 vs 21.1%). We then analyzed the costimulatory surface markers expression of DPDC and SPDC subsets. As shown in Fig. 1,B, compared with SPDC, a higher proportion of DPDC expressed CD80 (57.9% (34.2/(34.2 + 24.9)) vs 37% (15.4/(15.4 + 25.5))) and CD86 (60.6% (36.1/(36.1 + 23.5)) vs 36.6% (14.4/(14.4 + 26.0))) molecules on their cell surface. Similar differences in CD80 (43.5 vs 38.5%) and CD86 (30.1 vs 9.2%) expression between DPDC and SPDC were found in uninfected mice, although the levels were lower than corresponding 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. 1 C, DPDC produced significantly higher levels of IL-12 and IL-10 than SPDC. Similar differences were found between the subsets of DCs from uninfected mice, although the absolute levels were lower than those in infected mice. These results indicate that CD8α⁺ DC from infected mice express higher level of costimulatory molecules and produce higher immune regulatory cytokines than CD8⁻ DC.

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 adoptive transfer. The overall physical condition of the mice that received 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 adoptive transfer of DC subsets from naive mice (CD11c⁺CD8α⁺ (nDPDC) or CD11c⁺CD8α⁻ (nSPDC)) had no significant effect on mice conditions. The analysis of chlamydial loads in the lung showed that the recipients of iDPDC had significantly lower (1000-fold less) in vivo chlamydial growth than the control mice treated with PBS only (Fig. 3). In correlation with the finding in body weight changes, the recipients of iSPDC also showed less chlamydial growth in the lung than control mice, but their chlamydial loads were still 100 times higher than the iDPDC recipients (Fig. 3). In contrast, the adoptive transfer of nDPDC or nSPDC failed to show protective effects (Figs. 2 and 3). In addition, the recipients of iDPDC showed most mild pathological changes in the lung compared with other groups of mice, as demonstrated by less cellular infiltration (Fig. 4). The results indicate that the adoptive transfer of either DC subsets from infected mice can induce significant protective immunity in the recipients, but that CD8α⁺ DC are much more potent in inducing protection than CD8α⁻ DC.

To examine the relationship between the degree of protection observed in mice delivered with different DC subsets and the types of 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 CD8α⁺ DC from iDPDC mice displayed markedly stronger DTH reaction than the other groups of mice. The data suggest that CD8α⁺ 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 CD8α⁺ DC from iDPDC mice compared with those treated with PBS (Fig. 6). The transfer of CD8α⁻ 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 reduction in iDPDC recipients (Fig. 6). Taken together, the results indicate that the type of immune responses, including DTH and the isotypes of Ab production, were altered in mice pretreated with DCs isolated from MoPn-infected mice following challenge infection. In particular, adoptive transfer of iDPDC increased DTH but decreased IgG1 and IgE Ab responses.

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 and draining lymph node cells from different groups of mice. The results revealed that both spleen and lymph node cells of MoPn-infected mice pretreated with iDPDC produced significantly higher levels of TNF-α, IFN-γ, and IL-12 compared with the mice without DC adoptive transfer (Fig. 7). Notably, the recipients of iSPDC also showed increased production of the Th1-related cytokines but not to the degree in the iDPDC recipients. Interestingly, the recipients of nDPDC also showed significant increase of TNF-α and IFN-γ, although they were lower than that in the recipients of DC subsets from infected mice. In contrast, Th2-like cytokines such as IL-4, IL-5, and IL-13 were significantly reduced in iDPDC-treated mice (Fig. 8). The transfer of iSPDC also appeared to decrease Th2 cytokine production but to a lesser degree. These results, together with the finding in the types of immune responses (DTH and Ab isotypes), demonstrate a powerful role of CD8α⁺ DC in promoting protective Th1-type immune responses to chlamydial infection.

In the present model, we have shown that the transfer of CD8α⁺ DC, compared with CD8α⁻ DC, isolated from C. trachomatis MoPn-infected mice are much more capable of transferring protective immunity to naive mice to subsequent challenge infection. To our knowledge, this is the first report showing variable capacity of DC subsets in inducing protective immunity against chlamydial infection. Moreover, our results showed that the DC subsets were different in costimulatory molecule expression and cytokine production. In particular, CD8α⁺ DC from infected mice secreted IL-12 and IL-10 at significantly higher levels compared with CD8α⁻ DC from the same mice or either subset isolated from naive mice. The ability of CD8α⁺ DC to produce higher levels of these cytokines indicates a pertinent role for these cytokines in the transfer of protective immunity against subsequent challenge infection.

The approach used in this study is more physiological than most previously reported studies on the function of DC subsets. Although there is no direct evidence showing the carrying of chlamydial Ags by the transferred DC, the fact that the sera collected from recipients of DC from infected mice without further challenge infection showed Ab to MoPn Ags by Western blot analysis (data not shown) indicated that the DC from infected mice carry chlamydial Ags and/or antigenic peptides. Since the transferred DC subsets from infected mice carried chlamydial Ags/peptides, a faster protective immunity can develop. At the same time, the difference in protection between CD8α⁺ and CD8α⁻ subsets from infected mice may reflect their difference of potency in inducing protective responses. Notably, most of the previous studies examined bone marrow-derived or peripheral monocyte-derived DC, which were manipulated in in vitro culture conditions to generate different DC subsets (45, 46). These subsets were further examined by performing coculture with T cells or adoptively transferred to recipients after pulsing with Ags (45). A few studies used splenic CD8α⁺ and CD8α⁻ DC subsets, in which the DC subsets were pulsed with Ags in vitro followed by adoptive transfer of the cells (46). Although these approaches are useful in testing the immunological function of various Ags, the handling of these Ags by DC subsets in vitro may not necessarily reflect the physiological process in vivo. In contrast, in the present study, the Ag-processing steps and the development of DC subsets occurred in vivo conditions following infection. Furthermore, these DC subsets were adoptively transferred to recipient mice immediately after isolation without any further culture or manipulation; these results thus arguably more mimicking the function of DC subsets in vivo. In fact, the conclusion generated from the current approach and other studies (36) is very consistent in that CD8α⁺ DC are more potent in inducing Th1-like responses than CD8α⁻ DC. The powerful Th1-inducing function of CD8α⁺ DC is further supported by the finding that adoptive transfer of CD8α⁺ DC from naive mice also increased IFN-γ and TNF-α production (Fig. 7), although this was not strong enough to alter the type of immune responses (Figs. 5 and 6) and the outcome of protection (Figs. 2 and 3). Since mice show significant increase of CD8α⁺ DC following MoPn infection (Fig. 1 A), one might argue the mice should be protected even without DC transfer. Indeed, we have reported previously that mice started to recover after MoPn infection at 13–15 days following intranasal infection even in the absence of DC transfer (17, 47). A major reason for the natural recovery of the mice is likely because of the significant increase of CD8α⁺ DCs and the subsequent development of protective adaptive immune responses.

Notably, although our data strongly suggest that CD8α⁺ DCs are DC1-like cells, which preferentially induce Th1-like responses, the CD8α⁻ DCs in our model are not typical DC2-like cells. In fact, the adoptive transfer of CD8α⁻ DCs from infected mice also enhanced Th1-related cytokine production (Fig. 7) and inhibited Th2-related cytokine production (Fig. 8) and generated certain degree of protection (Figs. 2 and 3). One possible reason for this is that the CD8α⁻ DCs are still a heterogeneous population, which includes both DC1- and DC2-like cells, and the DC1-like subset is again predominant. Indeed, we found that within the CD8α⁻ DC population, further separation based on CD4 expression could obtain distinct CD4⁺ and CD4⁻ DC subsets, and the former produced least amount of IL-12 in the in vitro culture compared with CD8α⁺CD4⁻ and CD8α-CD4⁻ DC (X. Han and X. Yang, unpublished data). Therefore, using CD8α as a sole discriminative marker for DC1- and DC2-like cells is at least an over simplicity of DC biology and function. It is very likely that heterogeneity of DC in vivo is far beyond our imagination.

The finding that different DC subsets induce different levels of protection may be helpful in understanding the previous reports on the role of DC in chlamydial immunobiology and opens the door for further immunological and vaccination studies. For example, Su et al. (37) and Shaw et al. (38) have reported that adoptive transfer of whole Chlamydia organism-pulsed bone marrow- derived DC induce strong Th1 responses and protection to challenge infection, while major outer membrane protein (MOMP)-pulsed bone marrow-derived DC induce Th2-type responses and failed to provide protection (48). In addition, these authors also found that the DC pulsed with whole chlamydial organism produce higher IL-12 than the DC pulsed with MOMP. Therefore, it is likely that the two systems preferentially elicit different DC subsets, i.e., the former mainly induce DC1-like cells that are functionally similar to the CD8α⁺ DC in the present study, thus inducing strong Th1 responses, while the latter prefer the development of DC2-like cells. Moreover, although the DC with CD8α marker may not necessarily be the cell to be focused on for future vaccine studies, our present finding suggests that it is very important to realize the potential development and involvement of various DC subsets following chlamydial infection or vaccination.

A rather surprising but also very interesting finding in the present study is the higher IL-10 (in addition to higher IL-12) production by CD8α⁺ DCs from infected mice, which confer strong protective immunity against chlamydial infection challenge. Although IL-12 has been consistently found to be associated with protection (28, 40), higher IL-10 production has been previously linked to susceptibility to C. trachomatis infection and immunopathology in mouse and human chlamydial diseases (17, 47, 49). It remains unclear why a DC subset, which produces higher levels of these two apparently functionally counteractive cytokines, showed dominant protective phenotype. However, there are several possibilities, which may account for this observation. First, it is possible that the relative level of production of these two cytokines by CD8α⁺ DCs are variable, leading to a higher IL-12:IL-10 ratio, thus enhancing Th1 responses. Indeed, our results showed that CD8α⁺ DCs produced 2.5-fold higher IL-10 and 7.5-fold higher IL-12 than CD8α⁻ DC. Therefore, it is likely that the relative degree of IL-12/IL-10 production, rather than the absolute value of IL-10 or IL-12, is more important in determining the type of immune response and protection. Since the recipients of CD8α⁺ DCs exhibit higher cell-mediated immunity (DTH) and lower IgE and IgG1 Ab production, the results further confirm the importance of Th1-type immunity in host defense against Chlamydia infection.

The finding that the approaches, which induce higher IL-12 as well as IL-10, can elicit strong protection is not without precedence in chlamydial studies. We have demonstrated in previous studies that DNA vaccination using the chlamydial MOMP gene (omp1) induces both IL-12 and IL-10 production but provides significant protection (50). Moreover, we have also shown that vaccination with live, compared with dead, chlamydial organisms induced both higher IL-12 as well as IL-10 but provided strong protection (41). Does higher IL-10 production by CD8α⁺ DC play any positive role in enhancing protection? Notably, the transfer of CD8α⁺ DC from infected mice not only induced significantly higher IFN-γ and TNF-α production but lowered organism-driven Th2 cytokine (IL-4, IL-and IL-13) production as well (Figs. 7 and 8). Recent studies have shown that IL-10 production by tolerogenic DC can induce T regulatory cells, which can inhibit Th2 cells in allergy models (51). Therefore, in addition to increase Th1 response by higher IL-12 production and higher IL-12:IL-10 ratio, CD8α⁺ DC may also promote protection by way of inhibiting Th2 cytokine production through IL-10 production. Furthermore, it may be also explained that IL-10 produced by the DC may play a role in further “fine tuning” the DC for optimal function during immune responses by promoting prolonged interactions of activated DC and T cells, which is required for effective T cell priming. Such activity may enhance the development of Th1 responses, which is ultimately important for protection against this pathogen. It is also likely that the CD8α⁺ DC population is still heterogeneous, meaning that some CD8α⁺ DC produce higher IL-12, thus DC1-like while others produce higher IL-10 and thus like tolerogenic DC, which may lead to unresponsiveness of Th2 cells or the development of regulatory T cells. However, recent studies suggest that there seems to be a high degree of plasticity in the capacity of DC to prime T cells and direct their functional differentiation (52). In general, we feel that the results in the present study showing lower Th2 cytokine production in CD8α⁺ DC recipients may be a combinational outcome of higher IL-12 production by these DC and subsequent higher IFN-γ production by Th1 cells, which indirectly inhibit Th2 cells and the indirect inhibitory effect of IL-10 on Th2 cell development through the induction of regulatory T cells. Further studies on the heterogeneity of CD8α⁺ DC and analyzing DC from IL-10 gene KO mice or blocking IL-10 production by DC using RNA interference techniques may be helpful for addressing this question.

Collectively, the findings of the present study suggest that CD8α⁺ DCs, in contrast to CD8α⁻ DCs, play a crucial role in mounting protective immune responses to active chlamydial infection. It would be important to further investigate the conditions and the chlamydial components, which could predominantly induce the DC subset(s) beneficial for preventive and therapeutic strategies for chlamydial diseases.

The authors have no financial conflict of interest.