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Dendritic Cells in the Induction of Protective and Nonprotective Anticryptococcal Cell-Mediated Immune Responses¹

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Dendritic cells (DC) can be divided into three subsets, Langerhans cells, myeloid DC (MDC), and lymphoid DC (LDC), based upon phenotypic and functional differences. We hypothesized that different DC subsets are associated with the development of protective vs nonprotective cell-mediated immune (CMI) responses against the fungal pathogen, Cryptococcus neoformans. To test this, mice were immunized with protective and/or nonprotective immunogens, and DC subsets in draining lymph nodes were assessed by flow cytometry. The protective immunogen (cryptococcal culture filtrate Ag-CFA), in contrast to the nonprotective immunogen (heat-killed cryptococci-CFA), the nonprotective immunogen mixed with the protective immunogen (cryptococcal culture filtrate Ag + heat-killed cryptococci-CFA), or controls, stimulated significant increases in total leukocytes, Langerhans cells, MDC, LDC, and activated CD4⁺ T cells in draining lymph nodes. The protective immune response resulted in significantly increased levels of anticryptococcal delayed-type hypersensitivity reactivity and activated CD4⁺ T cells at the delayed-type hypersensitivity reaction site. Draining lymph node LDC:MDC ratios induced by the protective immunogen were significantly lower than the ratios induced by either immunization in which the nonprotective immunogen was present. In contrast, mice given the nonprotective immunogen had LDC:MDC ratios similar to those of naive mice. Our data indicate that lymph node Langerhans cells and MDC are APC needed for induction of the protective response. The predominance of LDC in mice undergoing nonprotective responses suggests that lymph node LDC, like splenic LDC, are negative regulators of CMI responses. In addition to showing DC subsets associated with functional differences, our data suggest that the LDC:MDC balance, which can be modulated by the Ag, determines whether protective or nonprotective anticryptococcal CMI responses develop.

	Introduction

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Since the first description of dendritic cells (DC)³ in 1973 (1), it has become increasingly apparent that they are a heterogeneous population of APC (2). Most investigations examining the phenotype in parallel with the function of DC have been performed on splenic DC despite the fact that most immune responses to infectious agents are initiated in draining lymph nodes (3, 4). Splenic DC can be divided phenotypically and functionally into two distinct populations, CD8^- and CD8⁺ DC. The CD8^- cells are myeloid DC (MDC), and the CD8⁺ cells are lymphoid DC (LDC) (5, 6, 7, 8, 9, 10). Although both populations can induce CD4⁺ T cell proliferation in vitro, MDC are better inducers of CD4⁺ T cell proliferation (7). An additional DC subset that is efficient at stimulating cell-mediated immune (CMI) responses is Langerhans cells, which are found in lymph nodes, but not spleens, of naive mice (11, 12). In contrast, LDC from the spleen play a role in maintenance of peripheral tolerance through induction of apoptosis in activated, Fas-expressing CD4⁺ T cells (7). Based on the present understanding of the functional characteristics of the DC subsets, a reasonable hypothesis is that the development of different types of immune responses involving CD4⁺ T cells, such as a protective vs a nonprotective response to an infectious agent, might be the result of the dominance of one DC subset over another during the induction phase of the response.

Cryptococcus neoformans is a yeast-like organism that causes frequently fatal meningitis in immunocompetent and immunocompromised humans (reviewed in Ref. 13). CMI responses against C. neoformans have been extensively studied (14, 15, 16, 17, 18). Because DC are the most effective APC for inducing CMI responses (19, 20, 21), they most likely have a critical role in the initiation of the anticryptococcal CMI response. Yet, the involvement of DC in CMI responses to C. neoformans has not been assessed. We have previously shown that one can induce either protective or nonprotective CMI responses to C. neoformans in mice depending on the immunogen (22). Immunization with the soluble cryptococcal culture filtrate Ag (CneF) emulsified in CFA induces activated CD4⁺ Th1 cells (23, 24) that provide protection against C. neoformans infection (22). In contrast, immunization with heat-killed cryptococci (HKC) in CFA induces different functionally defined T cell populations (24, 25, 26, 27) that do not provide protection (22) and can even exacerbate the disease (28) (J. W. Murphy, unpublished observations). Both immunizations induce anticryptococcal CMI responses detectable by footpad swelling, although the response induced by HKC-CFA is generally half that induced by CneF-CFA (27).

The present studies used a murine immunization model to test whether different subsets of DC are associated with protective vs nonprotective CMI responses to C. neoformans Ags. Our data show that MDC and Langerhans cells are increased in draining lymph nodes undergoing the protective CMI response. By comparison, LDC are the predominant APC present during induction of the nonprotective anticryptococcal CMI response. Moreover, the protective response led to increased numbers of activated CD4⁺ T cells in the draining lymph nodes and at an anticryptococcal DTH reaction site, whereas the nonprotective response did not. The addition of the nonprotective immunogen to the protective immunogen (CneF+HKC-CFA) resulted in 1) similar DC populations in the draining lymph nodes as observed in HKC-immunized mice, 2) reduced numbers of activated CD4⁺ T cells in the draining lymph nodes and at the anticryptococcal DTH reaction site compared with the protective immunogen, 3) a reduction in the magnitude of the anticryptococcal DTH response compared with the protective immunogen, and 4) a reduction in the survival time after challenge with C. neoformans compared with the protective immunogen. These combined data indicate that the DC subset balance relates to the type of CMI response (protective vs nonprotective) that develops in the draining lymph nodes during immune response induction and that the Ag can modulate this balance.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facility at the University of Oklahoma Health Sciences Center. Three to five mice per group were used in all experiments except the survival experiment, which had 10–20 mice/group. The mice were 7–10 wk of age at the beginning of each experiment.

Maintenance of endotoxin-free conditions

All reagents for injection into animals were tested for endotoxin content and were not used if the endotoxin level was detectable by the Limulus amebocyte chromogenic assay (BioWhittaker, Walkersville, MD; minimal detectable level of endotoxin, 0.1 ng endotoxin/ml). Sterile tissue culture plasticware was used whenever possible. All glassware was baked at 180°C for 3 h to destroy endotoxin.

Cryptococcal Ags, immunization, and infection

CneF from C. neoformans 184A was prepared as previously described (23). HKC were prepared by incubating C. neoformans isolate 184A for 1 h at 60°C (26). Mice were injected s.c. at two sites at the base of the tail with 0.2 ml of a 1:1 emulsion of CneF in CFA, sterile physiological saline in CFA, 10⁷ HKC in CFA, or CneF plus 10⁷ HKC in CFA. As controls for each immunization, five additional mice were immunized to assess the level of delayed-type hypersensitivity (DTH) responsiveness induced. For survival studies, 7 days after immunization mice were injected i.v. with 9 x 10⁴ viable 184A C. neoformans cells in 0.2 ml of saline, and the percent survival was followed.

Detection of anticryptococcal DTH responsiveness

At 7 days after immunization, the hind footpads of the mice were measured and then injected with 30 µl of saline in the left footpad and 30 µl of CneF in the right footpad. The footpads were measured 24 h after the challenge injection. The increase in footpad thickness was calculated by subtracting the difference in swelling in the 0 and 24 h measurements of the saline-injected footpad from the difference in swelling between the 0 and 24 h measurements of the CneF-injected footpads.

Preparation of single-cell suspensions

Lymph nodes (superficial inguinal, lumbar, and popliteal) were removed at the indicated times and pushed through a wire mesh screen to prepare single-cell suspensions. The screens and cell suspensions were incubated for 30 min on ice in 100 U/ml collagenase D (Roche, Indianapolis, IN) in HBSS/5% FCS. The cell suspensions were washed and resuspended in HBSS/5% FCS. This method of DC isolation was selected because the procedure extracts all subsets of DC (9, 11). Furthermore, we avoided culture steps that might change the DC surface to a more activated phenotype.

Sponge implantation and injection with Ag

Gelatin sponges (Gelfoam sterile absorbable gelatin sponge, Upjohn, Kalamazoo, MI) were surgically implanted under aseptic conditions (23). Briefly, sponges were cut into 17 x 18 x 10-mm blocks before rehydrating with sterile HBSS containing 100 U of penicillin/ml and 100 mg of streptomycin/ml. Three days after immunization with CneF-CFA, HKC-CFA, CneF+HKC-CFA, or control treatment, mice were anesthetized, and sponges were implanted s.c. through an incision on the animals’ shaved backs. The incisions were closed with wound clips. Four days after implantation, one sponge was injected with 0.1 ml of CneF, and the other was injected with 0.1 ml of saline. The sponges were removed at 24 h after injection.

Sponge retrieval and disaggregation

Mice were euthanized before surgical removal of sponges. Sponges were put in Stomacher bags (Tekmar, Cincinnati, OH) with enzyme cocktail (400 U of collagenase/ml; Sigma, St. Louis, MO), then homogenized with three 10-s pulses on a Stomacher 80 Lab Blender (Tekmar) at 15-min intervals (29). During 15-min intervals, the sponge homogenates were incubated at 37°C. Following the disaggregation step, the sponge homogenates were filtered through 390-µm pore size nylon screens followed by passage through 140-µm pore size nylon screens and washed with HBSS. The erythrocytes in the sponge homogenates were lysed by treatment with Tris-NH₄Cl (17 mM Tris and 139.7 mM NH₄Cl), and the remaining cells were washed once with HBSS. Viable cell counts were made using trypan blue dye exclusion and a hemacytometer.

Flow cytometric analysis

Portions of the lymph node single-cell suspensions from individual mice (three mice per group) were immunolabeled with the designated mAbs. For the isotype control, another portion of each cell suspension was pooled within a treatment group before undergoing the staining procedure. Individual cell suspensions and pooled cell suspensions were treated with anti-CD16/32 (HB197, American Type Culture Collection, Manassas, VA) for 30 min at 4°C to block Fc receptors and thereby reduce nonspecific staining of cells. After centrifugation, cells were incubated for 30 min at 4°C in wash buffer (PBS, 0.1% NaN₃, and 0.1% BSA) containing fluorochrome-labeled mAbs or isotype control mAbs. After washing, the cells were fixed with 1% paraformaldehyde. The mAbs used in these studies included DEC205-FITC (NLDC-145, American Type Culture Collection), 33D1 (American Type Culture Collection) visualized by goat anti-rat-PE (Caltag, South San Francisco, CA), CD11c (N418, American Type Culture Collection) visualized by goat anti-hamster-PE (Caltag), CD8-biotin (CT-CD8a, Caltag) visualized by ultraAvidin-APC (Leinco, Ballwin, MO), CD11b-TriColor (M1/70, Caltag), CD40-PE (3/23, Caltag), CD80-PE (RMMP-2, Caltag), CD86-PE (GL1, PharMingen), I-A^k-PE (14V.18, Caltag), FasL-PE (MFL3, PharMingen), CD4-TriColor (CT-CD4, Caltag), CD45R/B220-biotin (RA3-6B2, Caltag), macrophage-TriColor (F4/80, Caltag), CD4-FITC (CT-CD4, Caltag), CD45RB-PE (16A, PharMingen), and fluorochrome-labeled isotype control mAbs (Caltag). After fixing, 100,000–250,000 cells were analyzed using a FACSCalibur flow cytometer in the core facility and WinMDI 2.8 software. The percentage of positive cells was the percentage of the designated cell population with fluorescent intensity above the fluorescent intensity of cells treated with isotype control mAbs. The total number of positive cells in each sample was determined by multiplying the percent positive cells for the sample by the total number of leukocytes in the sample, which was assessed by hemocytometer counts.

Statistical analysis

Means, SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest were used to analyze the data. Survival data were analyzed by Kaplan-Meier survival statistics. Groups were considered statistically different if p 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lymph node cells from naive CBA/J mice were divided into four major populations based on forward scatter (FSC) and DEC205 labeling

Lymph node cells from naive mice immunolabeled with the DEC205 mAb were analyzed by flow cytometry for fluorescence and FSC (size). Four distinct populations of cells were observed; they were, DEC205^-FSC^low (63%), DEC205^highFSC^low (30%), DEC205^highFSC^high (2.6%), and DEC205^lowFSC^high (1.7%; Fig. 1). The DEC205^-FSC^low population was 78% CD4⁺ T cells and 8% B cells (B220⁺) and was negative for F4/80 (macrophage marker). The DEC205^highFSC^low population was 95% CD8⁺ T cells and 8% F4/80⁺ cells. As previously defined by Vremec et al. (9) and Salomon et al. (11), DEC205^highFSC^high and DEC205^lowFSC^high cells were considered to be two distinct populations of DC. The DEC205^highFSC^high and DEC205^lowFSC^high cells combined made up 5% of lymph node cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Identification of four distinct populations of cells in the lymph nodes of naive mice. After collagenase digestion of lymph nodes, cells were stained with the mAb DEC205 and analyzed by flow cytometry. Cells (100,000–250,000 events were collected) were analyzed for DEC205 expression and FSC (size). Percentages are of total cells. Results are representative of five independent experiments with similar results.

By gating on DEC205^highFSC^high cells and analyzing for CD8 expression, we found that the DEC205^highFSC^high population of cells could be divided further into two subsets, CD8^low (M1) and CD8^high (M2; Fig. 2A). These subsets could also be divided based on their expression of MHC class II (Table I). The DEC205^highFSC^highCD8^low cells were MHC class II^high, and the DEC205^highFSC^highCD8^high cells were MHC class II^int (Table I). Vremec and Shortman (9) found the expression of CD11b and CD8 on splenic DC to be almost inversely related; however, the markers were not completely mutually exclusive. When we analyzed DC subsets for CD11b expression, we found that most DEC205^highFSC^highCD8^low cells (M1) were CD11b⁺ (78% positive; Fig. 2B), whereas about half of DEC205^highFSC^highCD8^high cells (M2) were CD11b^low (47%; Fig. 2C). There was a broad range of expression levels of CD11b on the DEC205^highFSC^highCD8^high cell population. The DEC205^lowFSC^high population was CD8^- (Fig. 2D) and mostly CD11b^low (Fig. 2E). Therefore, based on cell surface staining patterns and the large size of the cells, the cell subsets, DEC205^highFSC^highCD8^lowCD11b⁺, DEC205^highFSC^high CD8^highCD11b^low, and DEC205^lowFSC^highCD8^-CD11b^low, were considered DC (9, 11, 30). Based on this, three distinct DC subsets from the lymph nodes of naive CBA/J mice can be identified by flow cytometry, myeloid DC (MDC, DEC205^highFSC^highCD8^low CD11b⁺), lymphoid DC (LDC, DEC205^highFSC^highCD8^high CD11b^low), and Langerhans cells (DEC205^lowFSC^highCD8^- CD11b^low). These definitions are in keeping with the observations described by others (9, 11) and were used in the investigations presented here for designating, MDC, LDC, and Langerhans cells.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Cell surface phenotype reveals three distinct lymph node DC populations. After collagenase digestion of lymph nodes, the cell surface phenotype of FSChigh cells was determined by three-color flow cytometric analysis. DEC205highFSChigh and DEC205lowFSChigh cells were gated based on DEC205 and FSC staining and were further analyzed for CD8 and CD11b expression by analyzing 100,000–250,000 events. Open histograms show isotype-matched control staining. Cells were gated on DEC205highFSChigh cells and analyzed for CD8 expression (A). M1, low density expression of CD8; M2, high density expression of CD8 (A). DEC205highFSChigh cells were gated based on CD8low (B) or CD8high (C) and were analyzed for CD11b expression. DEC205lowFSChigh cells were analyzed for CD8 (D) and CD11b (E) expression. Results are representative of five independent experiments with similar results.

Cell surface receptors, such as MHC class II, CD40, CD80, and CD86, which are necessary for effective Ag presentation to T cells, were assessed in each DC population. All three DC populations expressed the DC markers, 33D1 and CD11c, and MHC class II, but at different levels, as indicated by the intensities of staining (Table I). MDC and Langerhans cells displayed high density expression of MHC class II, whereas LDC had an intermediate level of MHC class II expression. MDC expressed a high density of CD86, with intermediate levels of CD40 and CD80. LDC and Langerhans cells displayed intermediate levels of CD86, but low levels of CD40 and CD80. To determine which DC population may be able to induce apoptosis, we also determined the levels of expression of FasL (CD95L) on the surface of cells in each population. LDC had more FasL than either MDC or Langerhans cells (Table I). MDC, LDC, and Langerhans cells comprised 18, 44, and 37%, respectively, of the FSC^high cells in the lymph nodes from naive CBA/J mice.

Kinetics of leukocyte influx into draining lymph nodes induced by protective and nonprotective cryptococcal immunizations

Before assessing the levels of various DC populations in the lymph nodes of mice immunized with C. neoformans Ags, we determined the total numbers of cells in draining lymph nodes. This was done by immunizing mice with the protective immunogen (CneF-CFA) or the nonprotective immunogen (HKC-CFA) or by treating mice with saline-CFA or nothing (naive) as controls. CneF-CFA-immunized mice had significantly more leukocytes in their draining lymph nodes than did HKC-CFA-immunized (p < 0.01), saline-CFA-treated (p < 0.05), or naive (p < 0.01) mice by 12 h after immunization (Fig. 3). The differences were even more pronounced by 18 h after immunization, when CneF-CFA-immunized mice had almost twice as many leukocytes as either HKC-CFA-immunized (p < 0.001) or saline-CFA-treated (p < 0.001) mice and 4 times as many leukocytes as naive mice (p < 0.001; Fig. 3). HKC-CFA-immunized mice (p < 0.001) and saline-CFA-treated (p < 0.001) mice had significantly more leukocytes than naive mice (Fig. 3). HKC-CFA-immunized and saline-CFA-treated mice had similar numbers of leukocytes in their draining lymph nodes at all times assessed (Fig. 3). Based on these data, the 18 h point was selected for additional experiments to gain an understanding of the cell populations in draining lymph nodes of mice undergoing the two different anticryptococcal CMI responses.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. Kinetics of leukocyte influx into draining lymph nodes in response to cryptococcal immunizations. Draining lymph nodes were removed at 3, 12, and 18 h from naive or saline-CFA-treated mice or mice immunized with CneF-CFA or HKC-CFA. After collagenase digestion, the number of leukocytes was assessed by hemocytometer counts. Results are representative of five independent experiments with similar results. Error bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (*, p < 0.05).

Because MDC and LDC from the spleen have been described to have stimulatory and regulatory functions, respectively, we were interested in determining whether the draining lymph node DC populations were differentially associated with protective vs nonprotective anticryptococcal CMI responses. We found that CneF-CFA-immunized and saline-CFA-treated mice had similar percentages of MDC (Fig. 4A) and similar percentages of LDC (Fig. 4B). In contrast, HKC-CFA-immunized mice had a significantly reduced percentage of MDC (Fig. 4A) and a significantly increased percentage of LDC (Fig. 4B) compared with saline-CFA-treated (p < 0.001) or CneF-CFA-immunized (p < 0.001) mice. Considering that the CneF-CFA-immunized mice had significantly more cells in their draining lymph nodes than control-treated or HKC-CFA-immunized mice, it is not surprising that the number of MDC (Fig. 4C) in CneF-CFA-immunized mice was significantly increased compared with that in saline-CFA-treated controls (p < 0.01) or HKC-CFA-immunized mice (p < 0.01). In addition, CneF-CFA-immunized mice had significantly more LDC (Fig. 4D) than saline-CFA-treated controls (p < 0.01) or HKC-CFA-immunized mice (p < 0.01). The percentages of MDC- and LDC-expressing costimulatory molecules (CD40, CD80, and CD86) were similar in immunized and control-treated mice (data not shown), whereas HKC-CFA-immunized mice had significantly higher percentages (p < 0.006) of LDC-expressing FasL (15.7 ± 0.8) than CneF-CFA-immunized (12.1 ± 0.5) or control-treated (12.3 ± 0.1) mice. The percentages of MDC-expressing FasL were similar in immunized and control-treated mice (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Effects of cryptococcal immunizations on lymph node MDC and LDC populations. Draining lymph nodes were removed from mice at 18 h after immunization with CneF-CFA or HKC-CFA or treatment with saline-CFA. After collagenase digestion, cells were stained for MDC and LDC as described previously, and 100,000–250,000 events were analyzed by three-color flow cytometric analysis. The percentages of MDC (A) and LDC (B) of DEC205highFSChigh cells present in draining lymph nodes are shown. The total number of MDC (C) and LDC (D) present in draining lymph nodes are indicated. Results are representative of five independent experiments with similar results. Error bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (*, p < 0.05).

In our immunization model it is possible that Langerhans cells may be taking up Ag because Ags were injected in close proximity to the epidermis. If this is happening, we would expect to see increased numbers of Langerhans cells in draining lymph nodes from CneF-CFA-immunized mice compared with those in HKC-CFA-immunized or control-treated mice. To assess this, the cells in draining lymph nodes from the various groups of mice were immunolabeled for Langerhans cells. We found that draining lymph nodes from CneF-CFA-immunized mice had significantly increased numbers of Langerhans cells compared with draining lymph nodes from saline-CFA-treated (p < 0.001) or HKC-CFA-immunized (p < 0.001) mice (Fig. 5), whereas HKC-CFA-immunized and saline-CFA-treated mice had similar numbers of Langerhans cells in their draining lymph nodes (Fig. 5). The percentages of Langerhans cells in draining lymph nodes were similar for all treatment groups, ranging from 40–50% of the FSC^high cells. Also, the percentages of Langerhans cells expressing costimulatory molecules (CD40, CD80, and CD86) and FasL were similar in immunized and control-treated mice (data not shown).

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Langerhans cell migration into draining lymph nodes in response to cryptococcal immunizations. Draining lymph nodes were removed from mice at 18 h after immunization with CneF-CFA or HKC-CFA or treatment with saline-CFA. After collagenase digestion, cells were stained for Langerhans cells as described previously, and 100,000–250,000 events were analyzed by three-color flow cytometric analysis. Results are representative of five independent experiments with similar results. Error bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (*, p < 0.05).

Considering that MDC have stimulatory function and their numbers were highest in the lymph nodes from CneF-CFA-immunized mice, we would predict that lymph nodes from those mice would have a greater number of CD4⁺ T cells with an activated phenotype (CD4⁺CD45RB^low) (31, 32) than would lymph nodes from HKC-CFA-immunized or control-treated mice. We stained draining lymph nodes from immunized and control groups of mice with mAbs to detect activation markers on CD4⁺ T cells. We found, as expected, that the lymph nodes from CneF-CFA-immunized mice had significantly more (p < 0.001) activated CD4⁺ T cells (1.33 ± 0.08 x 10⁶) than those from HKC-CFA-immunized (0.80 ± 0.04 x 10⁶), saline-CFA-treated (0.75 ± 0.07 x 10⁶), or naive (0.35 ± 0.02 x 10⁶) mice. When CD4⁺ T cells were analyzed for CD69 expression, which is another activation marker for CD4⁺ T cells (33), we found that there were more CD4⁺ T cells that were CD69⁺ in CneF-CFA-immunized mice than in any other group (data not shown).

LDC:MDC ratio in draining lymph nodes of mice undergoing protective and nonprotective anticryptococcal CMI responses

The draining lymph nodes from CneF-CFA-immunized mice also had high numbers of LDC. In fact, CneF-CFA-immunized mice had higher numbers than HKC-CFA-immunized or control-treated mice. Based on data derived using splenic DC, it has been postulated that if both MDC and LDC present the same Ag, then Ag presentation by MDC is dominant over presentation by LDC (34). If this were true, then as the ratio between LDC and MDC is reduced, one would expect an increase in effective Ag presentation. In addition to the absolute numbers of incoming DC, the ratio of LDC:MDC also may be an important factor in determining the developmental pathway of T cells. With this in mind we calculated the LDC:MDC ratios for each of the experimental groups. Our prediction proved to be true. Mice undergoing induction of a protective anticryptococcal CMI response (CneF-CFA immunized) displayed a significantly reduced LDC:MDC ratio compared with naive mice (p < 0.001) or HKC-CFA-immunized mice (p < 0.001; Fig. 6). Further support was provided by the saline-CFA control group. Mice treated with saline-CFA develop an anti-Mycobacterium CMI response (35), and their LDC:MDC ratio was significantly reduced compared with that in naive mice (p < 0.001). The LDC:MDC ratio in the saline-CFA group was equivalent to the reduced ratio in the CneF-CFA group (Fig. 6). In contrast, the mice immunized with the nonprotective immunogen (HKC-CFA) had an LDC:MDC ratio that was not significantly reduced from the ratio in naive mice.

View larger version (17K): [in this window] [in a new window]   FIGURE 6. LDC:MDC ratio. The total number of LDC was divided by the total number of MDC from draining lymph nodes of immunized or control mice. Results are representative of five independent experiments with similar results. Error bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (*, p < 0.05).

Influence of HKC on activation of CD4⁺ T cells in draining lymph nodes induced by CneF-CFA

As we had observed earlier, there were significant increases in the numbers of activated CD4⁺ T cells in the draining lymph nodes from CneF-CFA-immunized mice compared with those in the lymph nodes from HKC-CFA-immunized (p < 0.001) or saline-CFA-treated (p < 0.001) mice (Fig. 7). Addition of HKC to the CneF-CFA immunogen (CneF+HKC-CFA) resulted in a significant reduction in the numbers of activated CD4⁺ T cells in the draining lymph nodes compared with numbers of activated CD4⁺ T cells in the draining lymph nodes of mice given CneF-CFA alone (p < 0.001). The total number of activated CD4⁺ T cells in the draining lymph nodes of the CneF+HKC-CFA group was similar to the total number of that cell population in the draining lymph nodes from HKC-CFA-immunized or saline-CFA-treated mice (Fig. 7).

View larger version (16K): [in this window] [in a new window]   FIGURE 7. Effects of cryptococcal immunizations on activation of CD4+ T cells in draining lymph nodes. Draining lymph nodes were removed from mice at 18 h after immunization with CneF-CFA, HKC-CFA, or CneF+HKC-CFA or treatment with saline-CFA. A total of 100,000–250,000 events were analyzed by two-color flow cytometric analysis for activated CD4+ T cells, identified as falling into the lymphocyte light scatter gate and CD4+CD45RBlow. Results are representative of at least three independent experiments with similar results. Error bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (*, p < 0.05).

Because fewer activated CD4⁺ T cells were produced in the draining lymph nodes of mice that were immunized with CneF+HKC-CFA than were produced in the draining lymph nodes of mice that received CneF-CFA, we hypothesized that the level of DTH reactivity in the Ag-injected footpad of the CneF+HKC-CFA-immunized mice would be reduced compared with the footpad responses in CneF-CFA-immunized mice. The data in Fig. 8 show that indeed mice immunized with the mixture of HKC and CneF-CFA expressed weaker anticryptococcal DTH reactivity than mice immunized with CneF-CFA alone (p < 0.001). The combined immunogens (CneF+HKC-CFA) induced anticryptococcal DTH responses that were significantly elevated over reactions in the HKC-CFA group (p < 0.001; Fig. 8).

View larger version (16K): [in this window] [in a new window]   FIGURE 8. DTH responsiveness induced by different cryptococcal immunogens. DTH responsiveness was elicited by CneF in mice immunized 8 days earlier with CneF-CFA, saline-CFA, HKC-CFA, or CneF+HKC-CFA. Results are representative of seven independent experiments with similar results. Error bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences. *, p < 0.001 for the CneF-CFA group compared with all other groups. **, p < 0.001 for the HKC-CFA group compared with the CneF+HKC-CFA and saline-CFA groups. ***, p < 0.001 for the CneF+HKC-CFA group compared with the saline-CFA group.

To further show that there is a direct relationship between the induction and expression phases of the anticryptococcal DTH response, we determined the numbers of activated CD4⁺ T cells that infiltrated into a DTH reaction site in mice that had received either CneF-CFA, HKC-CFA, or the combined immunogen. The prediction was that when more activated CD4⁺ T cells are produced in the draining lymph nodes, there should be more activated CD4⁺ T cells at the DTH reaction site. To assess this we used a sponge model. Sponges implanted in immunized mice when injected with the recall Ag act as surrogate DTH reaction sites and allow one to study the cells and cytokines involved in the expression or efferent phase of an anticryptococcal CMI response (23, 36, 37, 38, 39). Gelatin sponges were implanted into the backs of immunized mice, and after a sufficient time (4 days) for the sponges to become vascularized, one sponge was injected with CneF, and the other sponge was injected with saline. Twenty-four hours after sponge injection the sponges were removed and disaggregated. The cells were immunolabeled and evaluated by flow cytometry. Previous studies using this sponge model have shown that mice immunized with the protective immunogen (CneF-CFA) have significantly more CD4⁺ Th1 cells than control-treated mice (saline-CFA) (23). In the present study, mice immunized with the protective immunogen (CneF-CFA) had significantly more activated CD4⁺ T cells (CD4⁺CD45RB^low at the site of an ongoing anticryptococcal DTH reaction than did HKC-CFA-immunized (p < 0.01), CneF+HKC-CFA-immunized (p < 0.05), or saline-CFA-treated (p < 0.01) mice (Fig. 9). In contrast, the numbers of activated CD4⁺ T cells at the anticryptococcal DTH reaction site in mice immunized with HKC-CFA or CneF+HKC-CFA or treated with saline-CFA were not significantly different from each other (Fig. 9). When CD4⁺ T cells were analyzed for CD62L (L-selectin) expression, which is lost on activated T cells (40), we found that there were more CD4⁺ T cells that were CD62L^- in CneF-CFA-immunized mice compared with any other group (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 9. Effects of protective and nonprotective anticryptococcal CMI responses on the influx of activated CD4+ T cells into an anticryptococcal DTH reaction site. Sponges implanted into control and immunized mice were injected with saline or CneF at 7 days after immunization, and 24 h after sponge challenge the leukocytes were obtained from the disaggregated sponges and immunolabeled for flow cytometry. A total of 125,000 events were analyzed by two-color flow cytometric analysis for activated CD4+ T cells, identified as falling into the lymphocyte light scatter gate and CD4+CD45RBlow. Results are represented as the mean increase in activated CD4+ T cells, which was calculated by subtracting the number of activated CD4+ T cells in the saline-injected sponge from the number of activated CD4+ T cells in the CneF-injected sponge. Results are representative of at least two independent experiments with similar results. Error bars represent SEMs, and ANOVA with the Newman-Keuls multiple comparison posttest was used to determine significant differences (*, p < 0.05).

Having observed that fewer activated CD4⁺ T cells were present at the anticryptococcal DTH reaction site in mice immunized with CneF+HKC-CFA than in mice immunized with only the protective immunogen (CneF-CFA), we predicted that mice immunized with CneF+HKC-CFA would have decreased survival times compared with mice that received the protective immunogen alone (CneF-CFA). Indeed, this is what we saw when we monitored survival over a 30-day period after immunizing mice and then infecting them 7 days after immunization i.v. with 9 x 10⁴ viable C. neoformans cells (Fig. 10). CneF-CFA-immunized mice survived significantly longer (mean survival time, 26.5 ± 1.2 days) than mice immunized with CneF+HKC-CFA (mean survival time, 21.9 ± 1.6 days; p < 0.02), HKC-CFA (mean survival time, 16.1 ± 0.4 days; p < 0.0001), or treated with saline-CFA (mean survival time, 14.6 ± 1.1 days; p < 0.0001). Mice immunized with CneF+HKC-CFA survived significantly longer than HKC-CFA-immunized (p < 0.0001) or saline-CFA-treated (p < 0.001) mice, whereas HKC-CFA-immunized and saline-CFA-treated mice had similar survival times. The results of this survival study with CneF-CFA-immunized, HKC-CFA-immunized, and saline-CFA-treated mice are similar to our previous observations (22).

View larger version (18K): [in this window] [in a new window]   FIGURE 10. Effects of protective and nonprotective cryptococcal immunogens on the survival of infected mice. Ten to 20 mice per group were immunized with CneF-CFA, HKC-CFA, or CneF+HKC-CFA or were treated with saline-CFA. Seven days after immunization the mice were infected i.v. with 9 x 104 viable 184A C. neoformans cells, and the percent survival was followed. Kaplan-Meier survival analysis was used to determine significant differences (*, p < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

With these ideas in mind, we studied the DC subsets in lymph nodes draining sites of immunization with cryptococcal Ags that induce either protective or nonprotective anticryptococcal CMI responses. The protective immunogen not only induced a significant increase in the total number of cells in the draining lymph nodes, it also preferentially induced more MDC and Langerhans cells that have the capability of presenting Ag and activating T lymphocytes. The ratio of LDC:MDC in the draining lymph nodes of the mice immunized with the protective immunogen compared with the LDC:MDC ratio in naive mice or mice immunized with the nonprotective immunogen was significantly reduced, indicating that the MDC had been preferentially increased. We must assume that the MDC and Langerhans cells in the draining lymph nodes performed their expected function because we also found a significant increase in CD4⁺ T cells with an activated phenotype in the lymph nodes draining the site of the protective immunogen. This same scenario was not true for the lymph nodes draining the site of immunization with the nonprotective immunogen. In the latter case, there was not a significant increase in the total numbers of cells, nor was the LDC:MDC ratio changed from that in lymph nodes from naive mice. In fact, immunization with the nonprotective immunogen resulted in significant increases over controls in the percentage of LDC in the lymph nodes, and concomitant with this, levels of activated CD4⁺ T cells did not exceed those in lymph nodes from control mice. The activated CD4⁺ T cells that were induced by the protective immunogen could be attracted to the site of cryptococcal Ag injection in the periphery, as shown by the increase in numbers of activated CD4⁺ T cells at the DTH reaction site in mice immunized with the protective immunogen. In mice given the nonprotective immunogen, the small numbers of activated CD4⁺ T cells produced in the lymph nodes resulted in low numbers of activated CD4⁺ T cells at the DTH reaction site.

By immunizing mice with the nonprotective immunogen mixed with the protective immunogen, we were able to shift the responses induced by the protective immunogen toward the direction of the response induced by the nonprotective immunogen. The shifts in the parameters were based, as would be predicted, on our interpretation presented above. The mixture of nonprotective and protective immunogens simultaneously affected several parameters. Addition of the nonprotective immunogen to the protective immunogen resulted in 1) down-regulation of the total numbers of cells in the draining lymph nodes, 2) inhibition of the reduction in the LDC:MDC ratio, 3) reduction in the numbers of activated CD4⁺ T cells in the draining lymph nodes as well as at the DTH reaction site, and 4) down-modulation of the protective immune response, as shown by the inability of mice immunized with the combination of immunogens to survive as long as mice immunized with the protective immunogen alone.

Based on the current understanding of cell-mediated immunity (3, 44) and our data, we propose a model for the induction of a protective anticryptococcal CMI response. The model consists of the following sequence of events: 1) Ag (CneF) with the adjuvant (CFA) cause the production of proinflammatory cytokines by resident tissue cells, establishing an inflamed microenvironment (reviewed in Refs. 12, 41 , and 45); 2) Langerhans cells take up Ag at the site of Ag deposition (i.e., epidermis and s.c. spaces) (reviewed in Refs. 12 and 41); 3) uptake of Ag and exposure to proinflammatory cytokines induce Langerhans cell to undergo maturation (i.e., increased MHC class II expression, increased costimulatory molecule expression, etc.) (reviewed in Refs. 12 and 41) with the acquisition of a phenotype similar to that of MDC (11); 4) concomitant with maturation, Langerhans cells would migrate to the lymph nodes via the afferent lymphatics (reviewed in Refs. 12 and 41); 5) once inside the draining lymph node, MDC (i.e., mature Langerhans cells) would present the acquired Ags to naive CD4⁺ T cells; 6) when naive CD4⁺ T cells recognize Ag in the class II MHC plus costimulatory molecules, they become activated and proliferate, then migrate out of the lymph nodes; and 7) APC would then probably die by apoptosis (46) to effectively terminate the induction of new CD4⁺ T cells.

Our data suggest that the nonprotective immunogen disrupts the induction of the protective CMI response at the level of maturation of Langerhans cells into MDC and their subsequent migration into the draining lymph nodes. This is supported by the fact that mice given the nonprotective immunogen either alone or with the protective immunogen have reduced numbers of Langerhans cells and MDC in their draining lymph nodes compared with mice given the protective immunogen alone. The inability of the nonprotective immunogen to induce increased numbers of Langerhans cells and MDC above control levels (saline-CFA-treated mice) suggests that at the site of Ag deposition the nonprotective immunogen has pleiotropic antagonistic effects on the maturation of Langerhans cells into MDC and the migration of Langerhans cells into draining lymph nodes. The mechanisms that possibly play a role in blocking maturation and trafficking of Langerhans cells are 1) lack of or reduced induction of proinflammatory cytokines at the site of Ag injection, 2) minimal levels of chemokine induction (47), 3) defective chemokine receptor expression (48), 4) HKC could be toxic to Langerhans cells, and/or 5) the possibility that NK cells interacting with HKC could result in direct suppression or elimination of Langerhans cells (49, 50, 51, 52, 53). Any of these mechanisms would ultimately result in inefficient maturation and migration of Langerhans cells and ultimately inefficient activation of CD4⁺ T cells. Further studies are required to define the mechanism(s) responsible for the reduced numbers of DC moving into the draining lymph nodes of mice injected with the nonprotective immunogen.

The differences in the immunogen’s ability to induce activated CD4⁺ T cells in the draining lymph nodes directly correlates with the differences seen in the levels of DTH responsiveness, the numbers of activated CD4⁺ T cells at the DTH reaction site, and the level of protection when mice are challenged with C. neoformans. In accordance with this, the protective immunogen induces higher concentrations of IFN- at the site of a DTH reaction than does the nonprotective immunogen (K. L. Nichols, S. K. Bauman, and J. W. Murphy, manuscript in preparation) or control treatments (23). Increased numbers of IFN--producing-activated CD4⁺ T cells lead ultimately to enhanced activation of natural effector cells (reviewed in Ref. 54). IFN--activated natural effector cells more efficiently kill C. neoformans (55, 56, 57), resulting in increased protection to the host upon subsequent infection. In fact, mice that received the protective immunogen survived significantly longer than mice that received either the nonprotective immunogen alone or the nonprotective immunogen mixed with the protective immunogen.

In summary, this is the first report of studies performed in vivo to associate function with the different DC populations in draining lymph nodes during the induction of CMI responses to a human pathogen. The data show that increases in stimulatory MDC and Langerhans cells in the lymph nodes draining the site of Ag deposition are needed to induce a protective anticryptococcal CMI response that functions to increase the number of activated CD4⁺ T cells in the draining lymph nodes, at an anticryptococcal DTH reaction site, and presumably at the infection sites. By comparison, high levels of regulatory LDC are associated with a nonprotective anticryptococcal CMI response and stimulate no measurable increase in numbers of activated CD4⁺ T cells. Thus, the balance of stimulatory vs regulatory DC has a profound effect on the developing CMI response. In addition, the nature of the Ag can affect the balance between these two disparate DC populations and, ultimately, whether a protective CMI response develops against C. neoformans.

	Acknowledgments

 
We greatly appreciate the assistance of Dr. Doug Drevets in the critical evaluation of this manuscript, Fredda Schafer for technical assistance with animal procedures, and Jim Henthorn for technical assistance with flow cytometric analyses. Flow cytometric analyses were performed at the Flow Cytometry and Cell Sorting Core Facility for Molecular Medicine and St. Francis Research Institute, University of Oklahoma Health Sciences Center.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grants AI15716, HL59852, and T32AI07364-07.

² Address correspondence and reprint requests to Dr. Juneann Murphy, Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, P.O. Box 26901, BMSB 1053, Oklahoma City, OK 73190.

³ Abbreviations used in this paper: DC, dendritic cells; CMI, cell-mediated immune; MDC, myeloid DC; LDC, lymphoid DC; CneF, cryptococcal culture filtrate Ag; HKC, heat-killed cryptococci; DTH, delayed-type hypersensitivity; FSC, forward scatter.

Received for publication November 5, 1999. Accepted for publication April 20, 2000.

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